Conversion of apoptotic proteins

ABSTRACT

Compounds that modulate the function of anti-apoptotic proteins such as Bcl-2 and Bcl-X L  are identified. These compounds have the ability to convert the activity of Bcl-2-family member proteins from anti-apoptotic to pro-apoptotic. Methods for inducing apoptosis are described, together with methods for identifying molecules that induce apoptosis through interaction with Bcl-2-family members.

RELATED APPLICATIONS

[0001] This application claims priority of United States ProvisionalApplication 60/433,535, filed Dec. 12, 2002, herein incorporated byreference in its entirety.

[0002] This invention was made in part with United States governmentsupport under grant numbers NIH CA60988, CA8700, and GM60554 awarded bythe National Institutes of Health, and USARMY PCRP-001590 and BC-001182awarded by the Department of Defense. The U.S. government has certainrights in this invention.

BACKGROUND OF THE INVENTION

[0003] 1. Field of the Invention

[0004] Compounds are provided herein that bind to Bcl-2-family memberpeptides and alter their apoptosis regulatory function. Morespecifically, the use of peptides expressed by the TR3 gene or chemicalcompounds that mimic the effects of TR3 to induce Bcl-2 or Bcl-X_(L) tohave a pro-apoptotic effect and to induce a conformational change, andthe use of the protein TCTP or chemical compounds that mimic the effectsof TCTP to induce Bcl-X_(L) to have a pro-apoptotic effect, aredescribed.

[0005] 2. Description of the Related Art

[0006] Apoptosis, also known as programmed cell death, is aphysiological process through which the body disposes of unneeded orundesirable native cells. The process of apoptosis is used duringdevelopment to remove cells from areas where they are no longerrequired, such as the interior of blood vessels or the space betweendigits. Apoptosis is also important in the body's response to disease.Cells that are infected with some viruses can be stimulated to undergoapoptosis, thus preventing further replication of the virus in the hostorganism.

[0007] Impaired apoptosis due to blockade of the cell death-signalingpathways is involved in tumor initiation and progression, sinceapoptosis normally eliminates cells with increased malignant potentialsuch as those with damaged DNA or aberrant cell cycling (White, 1996,Genes Dev, 10:1-15). The majority of solid tumors are protected by atleast one of the two cell death antagonists, Bcl-2 or Bcl-X_(L). Membersof the Bcl-2-family are known to modulate apoptosis in different celltypes in response to various stimuli. Some members of the family act toinhibit apoptosis, such as Bcl-2 and Bcl-X_(L), while others, such asBAX, BAK, Bid, and Bad, promote apoptosis. The ratio at which theseproteins are expressed can decide whether a cell undergoes apoptosis ornot. For instance, if the Bcl-2 level is higher than the BAX level,apoptosis is suppressed. If the opposite is true, apoptosis is promoted.Bcl-2 overexpression contributes to cancer cell progression bypreventing normal cell turnover caused by physiological cell deathmechanisms, and has been observed in a majority of cancers (Reed, 1997,Sem Hematol, 34:9-19; Buolamwini, 1999, Curr Opin Chem Biol, 3:500-509).The expression levels of Bcl-2 proteins often correlate with resistanceto a wide spectrum of chemotherapeutic drugs and γ-radiation therapy.Paradoxically, high levels of Bcl-2 also associate with favorableclinical outcomes for patients with some types of cancers. Therefore,Bcl-2 represents an excellent target for the treatment of cancer,especially those in which Bcl-2 is overexpressed and for whichtraditional therapy has failed.

[0008] Biological approaches targeted at Bcl-2 using antisenseoligonucleotides have been shown to enhance tumor cell chemosensitivity.Bcl-2 antisense oligonucleotides in combination with chemotherapy arecurrently in phase II/III clinical trials for the treatment of patientswith lymphoma and malignant melanoma, and further trials with patientswith lung, prostate, renal, or breast carcinoma are ongoing or planned(Reed, 1997, supra; Piche et al., 1998, Cancer Res 2134-2140; Webb etal., 1997, Lancet, 349:1137-1141; Jansen et al., 1998, Nat Med4:232-234; Waters et al., 2000, J Clin Oncol 18:1812-1823). Recently,cell-permeable Bcl-2 binding peptides and chemical inhibitors thattarget Bcl-2 have been developed, and some of them have been shown toinduce apoptosis in vitro and in vivo (Finnegan et al., 2001, Br JCancer 85:115-121; Enyedy et al., 2001, J Med Chem 44:4313-4324; Tzunget al., 2001, Nat Cell Biol 3:183-191; Degterev et al., 2001, Nat CellBio 3:173-182).

[0009] One well-established apoptotic pathway involves mitochondria(Green and Reed, 1998, Science 281: 1309-1312; Green and Kroemer, 1998,Trends Cell Biol 8: 267-271). Cytochrome c is exclusively present inmitochondria and is released from mitochondria in response to a varietyof apoptotic stimuli. Many Bcl-2-family proteins reside on themitochondrial outer membrane. Bcl-2 prevents mitochondrial disruptionand the release of cytochrome c from mitochondria, while BAX and BAKcreate pores in mitochondrial membranes and induce cytochrome c release.Recent evidence has indicated, however, that Bcl-2 under certainconditions can function as a pro-apoptotic molecule (Finnegan et al.,2001, supra; Fujita et al., 1998, Biochem Biophys Res Commun246:484-488; Fadeel et al., 1999, Leukemia 13:719-728; Grandgirard etal., 1998, EMBO J 17:1268-1278; Cheng et al., 1997, Science278:1966-1968; Del Bello et al., 2001, Oncogene 20:4591-4595). Bcl-2 canbe cleaved by caspase-3 and thus be converted to a pro-apoptotic proteinsimilar to BAX (Cheng et al., 1997, supra). Conversely, BAX has alsobeen shown to inhibit neuronal cell death when infected with Sinbisvirus (Lewis et al., 1999, Nat Med 5:832-835). These observationssuggest that members of the Bcl-2-farnily have reversible roles in theregulation of apoptosis and have the potential to function either as apro-apoptotic or anti-apoptotic molecule.

[0010] Members of the Bcl-2-family of proteins are highly related in oneor more specific regions, commonly referred to as Bcl-2 homology (BH)domains. BH domains contribute at multiple levels to the function ofthese proteins in cell death and survival. The BH3 domain, anamphipathic α-helical domain, was first delineated as a stretch of 16amino acids in Bak that is required for this protein to heterodimerizewith anti-apoptotic members of the Bcl-2-family and to promote celldeath. All proteins in the Bcl-2-family contain a BH3 domain, and thisdomain can have a death-promoting activity that is functionallyimportant. The BH3 domain acts as a potent “death domain” and there is afamily of pro-apoptotic proteins that contain BH3 domains which dimerizevia those BH3 domains with Bcl-2, Bcl-X_(L) and other anti-apoptoticmembers of the Bcl-2 family. Structural studies revealed the presence ofa hydrophobic pocket on the surface of Bcl-X_(L) and Bcl-2 that bindsthe BH3 peptide. Interestingly, the anti-apoptotic proteins Bcl-X_(L)and Bcl-2 also possess BH3 domains, but in these anti-apoptoticproteins, the BH3 domain is buried in the core of the protein and notexposed for dimerization. (Kelekar and Thompson, 1998, Trends Cell Biol8:324). NMR structural analysis of the Bcl-X_(L)/Bak BH3 peptide complexshowed that the Bak BH3 domain binds to the hydrophobic cleft formed inpart by the BH1, BH2 and BH3 domains of Bcl-X_(L) (Sattler, 1997,Science 275:983; Degterev, 2001, Nature Cell Biol 3:173-182).BH3-domain-mediated homodimerizations and heterodimerizations have a keyrole in regulating apoptotic functions of the Bcl-2-family (Diaz et al.,1997, J Biol Chem 272:11350; Degterev, 2001, Nature Cell Biol3:173-182).

[0011] The orphan receptor TR3 (also known as nur77 or nerve growthfactor-induced clone B NGFI-B) (Chang and Kokontis, 1988, BiochemBiophys Res Commun 155:971; Hazel et al., 1988, Proc Natl Acad Sci USA85:8444) functions as a nuclear transcription factor in the regulationof target gene expression (Zhang and Pfahl, 1993, Trends Endocrinol.Metab. 4:156-162; Tsai and O'Malley, 1994, Annu Rev Biochem 63:451;Kastner et al., 1995, Cell 83:859; Mageldorf and Evens, 1995, Cell83:841). TR3 was originally isolated as an immediate-early gene rapidlyexpressed in response to serum or phorbol ester stimulation of quiescentfibroblasts (Hazel et al., supra; Ryseck, et al., 1989, EMBO J 8:3327;Nakai et al., 1990, Mol Endocrinol 4:1438; Herschman, 1991, Annul RevBiochem. 60:281). Other diverse signals, such as membrane depolarizationand nerve growth factor, also increase TR3 expression (Yoon and Lau,1993, J Biol Chem 268:9148). TR3 is also involved in the regulation ofapoptosis in different cell types (Woronicz et al., 1994, Nature367:277; Liu et al., 1994, Nature 367:281; Weih et al., Proc Natl AcadSci USA 93:5533; Chang et al., 1997, EMBO J 16:1865; Li et al., 1998,Mol Cell Biol 18:4719; Uemura and Chang, 1998, Endocrinology 129:2329;Young et al., 1994, Oncol.Res. 6:203). It is rapidly induced duringapoptosis of immature thymocytes and T-cell hybridomas (Woronicz et al.,supra; Liu et al., supra), in lung cancer cells treated with thesynthetic retinoid 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalenecarboxylic acid (AHPN) (Li et al., supra) (also called CD437), and inprostate cancer cells treated with different apoptosis inducers (Uemuraand Chang, supra; Young et al., supra). Inhibition of TR3 activity byoverexpression of dominant-negative TR3 or its antisense RNA inhibitsapoptosis, whereas constitutive expression of TR3 results in massiveapoptosis (Weih et al., supra; Cheng et al., supra).

[0012] Further studies of TR3 have yielded a better understanding of itsmechanism of action in apoptosis (Li et al., 2000, Science 289:1159).First, several apoptosis inducing agents which also induced TR3expression in human prostate cancer cells were identified. Theseincluded the AHPN analog6-[3-(1-adamantyl)-4-hydroxyphenyl]3-chloro-2-naphthalenecarboxylic acid(MM11453), the retinoid(Z)-4-[2-bromo-3-(5,6,7,8-tetrahydro-3,5,5,8,8-pentamethyl-2-naphthalenyl)propenoyl]benzoicacid (MM11384), the phorbol ester 12-O-tetradecanoyl phorbol-13-acetate(TPA), the calcium ionophore A23187, and the etoposide VP-16. Second, itwas found that the transactivation activity of TR3 is not required forits role in inducing apoptosis, as demonstrated by an experiment thatshowed that apoptosis inducing agents blocked the expression of a TR3target reporter gene. This was further supported by the finding that aTR3 mutant deprived of its DNA binding domain (DBD) was still competentfor inducing apoptosis. Third, TR3 was found to relocalize to the outersurface of the mitochondria in response to some apoptotic stimuli. TR3,visualized in vivo by tagging with Green Fluorescent Protein (GFP), wasshown to relocalize from the nucleus to the mitochondria in response toapoptosis-inducing agents. Fractionation studies showed that TR3 wasassociating with the mitochondria-enriched heavy membrane fraction, andproteolysis accessibility studies on purified mitochondria confirmedthat TR3 was associating with the outer surface of the mitochondria,where Bcl-2-family members are also found. Fourth, TR3 was show to beinvolved in the regulation of cytochrome c release from themitochondria. Inhibition of TR3 activity by expression of TR3 antisenseRNA blocked the release of cytochrome c and mitochondrial membranedepolarization in cells stimulated with TPA and MM11453. Furthermore,incubating purified mitochondria with recombinant TR3 protein resultedin cytochrome c release.

[0013] Li et al., 2000, supra, further explored the function of TR3through mutation of the protein. A TR3 mutant which had the DNA bindingdomain (amino acid residues 168-467) removed (TR3/ΔDBD) no longerlocalized in the nucleus in non-stimulated cells, but instead wasconsistently found in mitochondria. This localization phenotype wasaccompanied by a constant release of cytochrome c from the mitochondria.Three other deletion mutants were also generated and assayed: anamino-terminal deletion of 152 amino acids referred to as TR3/Δ1, a 26amino acid carboxy-terminal deletion referred to as TR3/Δ2, and a 120amino acid carboxy-terminal deletion referred to as TR3/Δ3. The TR3/Δ1protein did not relocalize to the mitochondria in response to TPA, butmaintained a nuclear localization. TR3/Δ1 had a dominant negativeeffect, preventing the relocalization of full-length TR3 to themitochondria and inhibiting apoptosis. Mitochondrial targeting was stillobserved in TR3/Δ2 protein expressing cells, but not in TR3/Δ3 proteincells in response to TPA treatment. These results indicated thatcarboxy-terminal and amino-terminal sequences are crucial formitochondrial targeting of TR3 and its regulation.

[0014] Experiments designed to alter the localization of TR3/ΔDBD byfusing it to various cellular localization signals showed that TR3 musthave access to the mitochondria in order to induce its pro-apoptoticeffect. When TR3/ΔDBD was fused to a nuclear localization sequence, aplasma membrane targeting sequence, or an ER-targeting sequence,TR3/ΔDBD was not targeted to the mitochondria and no induction ofcytochrome c release was observed.

[0015] The translationally controlled tumor-associated protein (TCTP) isconserved across a wide range of eukaryotes and shows no significantsequence homology with any other proteins. The precise function of thefamily remains elusive. TCTP has been described as growth relatedprotein. TCTP was originally identified as a serum-inducible 23-kDaprotein band that undergoes an early and prominent increase upon serumstimulation in tissue culture cells (Benndorf et al., 1988, Exp CellRes174:130). TCTP mRNA is expressed at constant levels in both growingand nongrowing cells, and the translation is regulated by itspolypyrimidine-rich 5′ untranslated region (Bohm et al., 1991, BiomedBiochim Acta 50:1193; 174:130). TCTP was shown to be one of the firstproteins to be induced in Ehrlich ascites tumor cells following mitoticstimulation (Bohm et al., 1989, Biochem Int 19:277) and has been foundto be amongst a small group of Schizosaccharomyces pombe proteins thatare repressed in response to conditions that arrest cell growth, such asammonium starvation (Bonnet C. et al., 2000, Yeast 16:23). TCTP wasrecently shown to be a tubulin-binding protein that dynamicallyinteracts with microtubules during the cell cycle. In addition, TCTPlevels in overexpressing cells were correlated with microtubulestabilization and reduced growth rate in vivo (Gachet et al., 1999, JCell Sci 112:1257). The expression of TCTP also appears to be regulatedat two distinct levels in response to the concentration of calcium indifferent cellular compartments. Whereas depletion of the ER storecauses an increase in TCTP MRNA abundance, increased cytosolic calciumconcentrations regulate gene expression at the post-transcriptionallevel (Xu et al., 1999, Biochem J, 342:683). The solution structure ofTCTP forms a structural superfamily with the Mss4/Dss4 family ofproteins, which bind to the GDP/GTP-free form of Rab proteins (membersof the Ras superfamily) and have been termed guanine nucleotide-freechaperones (Thaw et al., 2001, Nat Struct Biol 8:701).

[0016] The identification of compounds having the ability to alter theactivity of Bcl-2-family members from anti-apoptotic to pro-apoptoticwould have important therapeutic applications, for example, in thetreatment of cancer and other diseases.

SUMMARY OF THE INVENTION

[0017] One aspect of the invention pertains to the discovery ofmolecules that modulate the activity of Bcl-2-family members in theirregulation of apoptosis. More specifically, it concerns regulators ofapoptosis which inhibit proteins such as Bcl-2 and Bcl-X_(L) and caninduce a conformational change in these proteins resulting inpro-apoptotic properties.

[0018] The scope of the compositions and methods described hereinincludes the use of proteins and their respective genes, peptides,peptide analogs, antibodies, polynucleotides and small molecules toregulate the apoptotic effect of Bcl-2-family members.

[0019] In one embodiment, a compound which binds to Bcl-2 and modulatesthe activity of Bcl-2 in a cell so as to be inductive of apoptosis,without cleaving Bcl-2, is described. The compound can be a peptide, apeptidomimetic, an antibody or a small organic molecule. In oneembodiment, the compound comprises TR3, whereas in another embodiment,the compound comprises the DC 1 region of TR3. In a further embodiment,the compound comprises an antibody which mimics the action of TR3 onBcl-2.

[0020] In another embodiment, a compound which binds to Bcl-X_(L) andmodulates the activity of Bcl-X_(L) in a cell so as to be inductive ofapoptosis, without cleaving Bcl-X_(L),is disclosed. The compound can bea peptide, peptidomimetic, or a small organic molecule. In oneembodiment, the compound comprises TCTP, whereas in another embodiment,the compound comprises a peptidomimetic which mimics TCTP.

[0021] Yet another embodiment of the invention is a method of inducingapoptosis in a mammalian cell, comprising contacting the cell with aneffective amount of a compound which binds to Bcl-2 and modulates theactivity of Bcl-2 so as to be inductive of apoptosis. Similarly, anotherembodiment includes a method of inducing apoptosis in a mammalian cell,comprising contacting the cell with an effective amount of a compoundwhich binds to Bcl-X_(L) and modulates the activity of Bcl-X_(L) in thecell so as to be inductive of apoptosis.

[0022] Another embodiment of the invention includes a method ofinhibiting apoptosis in a mammalian cell, comprising contacting the cellwith an effective amount of a compound that prevents the binding of TR3and Bcl-2. Another embodiment includes a method of inhibiting apoptosisin a mammalian cell, comprising contacting the cell with an effectiveamount of a compound that prevents the binding of TCTP and Bcl-X_(L).Another embodiment includes a method of identifying molecules thatinhibit apoptosis by preventing binding of TR3 to Bcl-2 or binding ofTCTP to Bcl-X_(L).

[0023] Still another embodiment of the invention includes a method ofidentifying molecules that induce apoptosis, comprising determining theability of said molecule to bind to a Bcl-2-family protein and modulatethe activity of said protein so as to be inductive of apoptosis.

BRIEF DESCRIPTION OF THE DRAWINGS

[0024]FIG. 1 shows the results of a reporter gene assay in CV-1 cells,which demonstrates the inhibition of TR3-dependent transactivation byBcl-2.

[0025]FIG. 2 is a schematic representation of TR3 mutants, indicatingthe DNA-binding and ligand-binding domains of TR3.

[0026]FIG. 3 is a schematic representation of Bcl-2 mutants with theBcl-2 homology (BH) and loop domains and α-helical region indicated.

[0027]FIGS. 4a-c are graphs showing that expression of both TR3/ΔDBD andBcl-2 results in apoptosis of lung cancer and breast cancer cells.

[0028]FIG. 5 is a graph showing the effect of TR3 mutants on apoptoticpotential of Bcl-2.

[0029]FIG. 6 is a graph showing that the BH3 domain is involved inTR3/ΔDBD-induced apoptosis.

[0030]FIG. 7 is a graph illustrating apoptosis in Bcl-X_(L)overexpressing cells induced by TCTP.

[0031]FIG. 8 is a graph that illustrates that the BH3 domain ofBcl-X_(L) is involved in TCTP induced apoptosis.

[0032]FIGS. 9a-f is a series of graphs that illustrate the Bcl-2 domainwhich undergoes a conformational change when TR3 binds.

DETAILED DESCRIPTION OF THE PREFERRED EMOBODIMENT I. Definitions andGeneral Parameters

[0033] The following definitions are set forth to illustrate and definethe meaning and scope of the various terms used to describe theinvention herein.

[0034] As used herein, “pharmaceutically or therapeutically acceptablecarrier” refers to a carrier medium which does not interfere with theeffectiveness of the biological activity of the active ingredients andwhich is not toxic to the host or patient.

[0035] As used herein, “stereoisomer” refers to a chemical compoundhaving the same molecular weight, chemical composition, and constitutionas another, but with the atoms grouped differently. That is, certainidentical chemical moieties are at different orientations in space and,therefore, when pure, have the ability to rotate the plane of polarizedlight. However, some pure stereoisomers may have an optical rotationthat is so slight that it is undetectable with present instrumentation.The compounds described herein may have one or more asymmetrical carbonatoms and therefore include various stereoisomers. All stereoisomers areincluded within the scope of the present invention.

[0036] As used herein, “therapeutically- or pharmaceutically-effectiveamount” as applied to the disclosed compositions refers to the amount ofcomposition sufficient to induce a desired biological result. Thatresult can be alleviation of the signs, symptoms, or causes of adisease, or any other desired alteration of a biological system. Forexample, the result can involve a decrease and/or reversal of cancerouscell growth.

[0037] As used herein, “homology” or “identity” or “similarity” refersto sequence similarity between two peptides or between two nucleic acidmolecules. Homology can be determined by comparing a position in eachsequence which may be aligned for purposes of comparison. When aposition in the compared sequence is occupied by the same base or aminoacid, then the molecules are identical at that position. A degree ofhomology or similarity or identity between nucleic acid sequences is afunction of the number of identical or matching nucleotides at positionsshared by the nucleic acid sequences. An “unrelated” or “non-homologous”sequence shares less than about 40% identity, though preferably lessthan about 25% identity, with one of the sequences described herein.

[0038] In addition to peptides consisting only of naturally-occurringamino acids, peptidomimetics or peptide analogs are also considered .Peptide analogs are commonly used in the pharmaceutical industry asnon-peptide drugs with properties analogous to those of the templatepeptide. These types of non-peptide compounds are termed “peptidemimetics” or “peptidomimetics” (see, e.g., Luthman et al., 1996, ATextbook of Drug Design and Development, 14:386-406, 2nd Ed., HarwoodAcademic Publishers; Grante, 1994, Angew Chem Int Ed Engl, 33:1699-1720;Fauchere, 1986, Adv Drug Res, 15:29; Evans et al., 1987, J Med Chem30:229, all of which are incorporated by reference). Peptide mimeticsthat are structurally similar to therapeutically useful peptides may beused to produce an equivalent or enhanced therapeutic or prophylacticeffect. Generally, peptidomimetics are structurally similar to aparadigm polypeptide (i.e., a polypeptide that has a biological orpharmacological activity), such as naturally-occurring receptor-bindingpolypeptide, but have one or more peptide linkages optionally replacedby a linkage selected from the group consisting of: —CH₂ NH—, —CH₂ S—,—CH₂—CH₂—, —CH═CH-(cis and trans), —COCH₂—, —CH(OH)CH₂—, and —CH₂ SO—,by methods known in the art and further described in the followingreferences: Spatola, 1983, In, Chemistry and Biochemistry of AminoAcids, Peptides, and Proteins, B. Weinstein, eds., Marcel Dekker, NewYork, p. 267; Hudson et al., 1979, Int J Pept Prot Res 14:177-185 (1979)(—CH₂ NH—, CH₂ CH₂—); Spatola et al., 1986, Life Sci 38:1243-1249 (—CH₂—S); Hann, 1982, J Chem Soc Perkins Trans I, 307-314 (—CH—CH—, cis andtrans); Almquist et al., 1980, J Med Chem 23:1392-1398 (—COCH₂—);Jennings-White et al., 1982, Tetrahedron Lett 23:2533 (—COCH₂—); Szelke,et al., European Appln. EP 45665 (1982) (—CH(OH)CH₂—); Holladay et al.,1983, Tetrahedron Lett 24:4401-4404 (—C(OH)CH₂—); and Hruby, 1982, LifeSci, 31:189-199 (—CH₂ —S—); each of which is incorporated herein byreference. A particularly preferred non-peptide linkage is —CH₂ NH—.Such peptide mimetics may have significant advantages over polypeptideembodiments, including, for example: more economical production, greaterchemical stability, enhanced pharmacological properties (half-life,absorption, potency, efficacy, etc.), altered specificity (e.g., abroad-spectrum of biological activities), reduced antigenicity, andothers. Labeling of peptidomimetics usually involves covalent attachmentof one or more labels, directly or through a spacer (e.g., an amidegroup), to non-interfering position(s) on the peptidomimetic that arepredicted by quantitative structure-activity data and/or molecularmodeling. Such non-interfering positions generally are positions that donot form direct contacts with the macromolecules(s) (e.g.,immunoglobulin superfamily molecules) to which the peptidomimetic bindsto produce the therapeutic effect. Derivitization (e.g., labeling) ofpeptidomimetics should not substantially interfere with the desiredbiological or pharmacological activity of the peptidomimetic. Generally,peptidomimetics of receptor-binding peptides bind to the receptor withhigh affinity and possess detectable biological activity (i.e., areagonistic or antagonistic to one or more receptor-mediated phenotypicchanges).

[0039] Systematic substitution of one or more amino acids of a consensussequence with a D-amino acid of the same type (e.g., D-lysine in placeof L-lysine) may be used to generate more stable peptides. In addition,constrained peptides comprising a consensus sequence or a substantiallyidentical consensus sequence variation may be generated by methods knownin the art (Rizo, et al., 1992, Annu Rev Biochem 61:387 (1992),incorporated herein by reference); for example, by adding internalcysteine residues capable of forming intramolecular disulfide bridgeswhich cyclize the peptide.

[0040] Synthetic or non-naturally occurring amino acids refer to aminoacids which do not naturally occur in vivo but which, nevertheless, canbe incorporated into the peptide structures described herein. Preferredsynthetic amino acids are the D-α-amino acids of naturally occurringL-α-amino acid as well as non-naturally occurring D- and L-α-amino acidsrepresented by the formula H₂ NCHR⁵ COOH where R⁵ is 1) a lower alkylgroup, 2) a cycloalkyl group of from 3 to 7 carbon atoms, 3) aheterocycle of from 3 to 7 carbon atoms and 1 to 2 heteroatoms selectedfrom the group consisting of oxygen, sulfur, and nitrogen, 4) anaromatic residue of from 6 to 10 carbon atoms optionally having from 1to 3 substituents on the aromatic nucleus selected from the groupconsisting of hydroxyl, lower alkoxy, amino, and carboxyl, 5)-alkylene-Ywhere alkylene is an alkylene group of from 1 to 7 carbon atoms and Y isselected from the group consisting of (a) hydroxy, (b) amino, (c)cycloalkyl and cycloalkenyl of from 3 to 7 carbon atoms, (d) aryl offrom 6 to 10 carbon atoms optionally having from 1 to 3 substituents onthe aromatic nucleus selected from the group consisting of hydroxyl,lower alkoxy, amino and carboxyl, (e) heterocyclic of from 3 to 7 carbonatoms and 1 to 2 heteroatoms selected from the group consisting ofoxygen, sulfur, and nitrogen, (f) —C(O)R² where R² is selected from thegroup consisting of hydrogen, hydroxy, lower alkyl, lower alkoxy, and—NR³ R⁴ where R³ and R⁴ are independently selected from the groupconsisting of hydrogen and lower alkyl, (g) —S(O)_(n) R⁶ where n is aninteger from 1 to 2 and R⁶ is lower alkyl and with the proviso that R⁵does not define a side chain of a naturally occurring amino acid.

[0041] Other preferred synthetic amino acids include amino acids whereinthe amino group is separated from the carboxyl group by more than onecarbon atom such as beta (β)-alanine, gamma (γ)-aminobutyric acid, andthe like.

[0042] Particularly preferred synthetic amino acids include, by way ofexample, the D-amino acids of naturally occurring L-amino acids,L-(1-naphthyl)-alanine, L-(2-naphthyl)-alanine, L-cyclohexylalanine,L-2-aminoisobutyric acid, the sulfoxide and sulfone derivatives ofmethionine (i.e., HOOC—(H₂ NCH)CH₂ CH₂ —S(O)_(n) R⁶) where n and R⁶ areas defined above as well as the lower alkoxy derivative of methionine(i.e., HOOC—(H₂ NCH)CH₂ CH₂ —OR⁶ where R⁶ is as defined above).

II. Overview

[0043] Compounds that bind to Bcl-2-family members and alter theirfunction in apoptosis are also provided. These compounds include “lead”peptide compounds and “derivative” compounds constructed so as to havethe same or similar molecular structure or shape as the lead compoundsbut that differ from the lead compounds either with respect tosusceptibility to hydrolysis or proteolysis and/or with respect to otherbiological properties, such as increased affinity for the receptor.

[0044] The examples described herein demonstrate that thenuclear-to-mitochondrial pathway of TR3 can be extended to lung andbreast cancer cells. In addition, it is shown that Bcl-2 acts as amitochondrial receptor of TR3 through their physical interaction. Inresponse to various apoptotic stimuli, the expression of the TR3 proteinis increased and its localization is altered from nuclear tocytoplasmic, more specifically to the outer member of mitochondria. Thisassociation with the mitochondria is the result of binding to the Bcl-2,whose normal function is the inhibition of apoptosis, particularly theinhibition of the release of cytochrome c from the mitochondria. Highexpression of TR3/ΔDBD (a form of TR3 without its DNA-binding domain)induces cytochrome c release and apoptosis only in cells expressingBcl-2, indicating that TR3 modulates the function of Bcl-2 fromanti-apoptotic to pro-apoptotic, without cleaving the Bcl-2 protein.Further data show that TR3 induces a conformational change in Bcl-2which may cause the function of Bcl-2 to be modified from ananti-apoptotic to a pro-apoptotic protein. Mutational analysis indicatedthat the C-terminal domain of TR3, which contains several α-helices, isresponsible for interacting with Bcl-2. A small fragment with only 69amino acids (DC1) is sufficient for interacting with Bcl-2 and inducingapoptotic potential of Bcl-2. When analyzing the Bcl-2 domains involvedin the interaction, it was observed that mutations in the hydrophobicpocket of Bcl-2 did not affect its interaction with TR3. Moreover, theN-terminal domain of Bcl-2, containing the loop region and BH4 domain,was able to interact with TR3/ΔDBD. Deletion of BH4 domain from Bcl-2did not affect the interaction of TR3/ΔDBD with Bcl-2, implying that theloop region of Bcl-2 was responsible for interaction. In addition, DC1and BH3-only Bcl-Gs did not compete for binding to Bcl-2. Instead,Bcl-Gs enhanced the binding of DC1 to Bcl-2. Thus, Bcl-2 was found tointeract with TR3 in a manner that is different from its interactionwith Bcl-2-family proteins containing only the BH3 domain.

[0045] Specific interaction of TR3 with Bcl-2 is essential for TR3 totarget mitochondria and results in conversion of Bcl-2 from ananti-apoptotic to a pro-apoptotic molecule. Concomitantly, theconformation of Bcl-2 is changed by TR3 resulting in the exposure of theotherwise hidden BH3 domain. Peptides derived from the specificBcl-2-interacting domain of TR3, such as DC1, will mimic its effect, aswill peptide analogs and small molecules designed to mimic the bindingproperties of the peptides. Further, antibodies may be identified whichalso mimic the effect of TR3. Peptides, antibodies, analogs, and smallmolecules that specifically interact with Bcl-2 will effectively induceapoptosis of cancer cells, thus restricting tumor growth. In addition,the results provide a molecular basis for developing various agents fortreating cancers and other therapeutic applications.

[0046] Similarly, TCTP was identified as another apoptosis regulatorymolecule which binds to an anti-apoptotic Bcl-2 related protein. Whileoverexpression of TCTP in cells induces apoptosis, this induction iseven more pronounced when the Bcl-X_(L) is co-expressed with it; likeTR3, TCTP is converting an anti-apoptotic protein to a pro-apoptoticprotein. Peptides, peptide analogs, and small molecules that mimic thebinding of TCTP to Bcl-X_(L) will also act to induce apoptosis.

III. Preparation of Peptides and Peptide Mimetics

[0047] A. Solid Phase Synthesis

[0048] The peptides disclosed herein can be prepared by classicalmethods known in the art, for example, by using standard solid phasetechniques. The standard methods include exclusive solid phasesynthesis, partial solid phase synthesis methods, fragment condensation,classical solution synthesis, and even recombinant DNA technology. See,e.g., Merrifield, 1963, J Am Chem Soc 85:2149, incorporated herein byreference. On solid phase, the synthesis is typically commenced from theC-terminal end of the peptide using an alpha-amino protected resin. Asuitable starting material can be prepared, for instance, by attachingthe required alpha-amino acid to a chloromethylated resin, ahydroxymethyl resin, or a benzhydrylamine resin. One suchchloromethylated resin is sold under the tradename BIO-BEADS SX-1™ byBio Rad Laboratories (Richmond, Calif.) and the preparation of thehydroxymethyl resin is described by Bodonszky et al., 1966, Chem Ind(London), 38:1597. The benzhydrylamine (BHA) resin has been described byPietta and Marshall, 1970, Chem Comm 650, and is commercially availablefrom Beckman Instruments, Inc. (Palo Alto, Calif.) in the hydrochlorideform.

[0049] Thus, the compounds disclosed herein can be prepared by couplingan alpha-amino protected amino acid to the chloromethylated resin withthe aid of, for example, a cesium bicarbonate catalyst, according to themethod described by Gisin, 1973, Helv Chim Acta 56:1467. After theinitial coupling, the alpha-amino protecting group is removed by achoice of reagents including trifluoroacetic acid (TFA) or hydrochloricacid (HCl) solutions in organic solvents at room temperature.

[0050] The alpha-amino protecting groups are those known to be useful inthe art of stepwise synthesis of peptides. Included are acyl typeprotecting groups (e.g., formyl, trifluoroacetyl, acetyl), aromaticurethane type protecting groups (e.g., benzyloxycarboyl (Cbz) andsubstituted Cbz), aliphatic urethane protecting groups (e.g.,t-butyloxycarbonyl (Boc), isopropyloxycarbonyl, cyclohexyloxycarbonyl)and alkyl type protecting groups (e.g., benzyl, triphenylmethyl). Bocand Fmoc are preferred protecting groups. The side-chain protectinggroup remains intact during coupling and is not split off during thedeprotection of the amino-terminus protecting group or during coupling.The side-chain protecting group must be removable upon the completion ofthe synthesis of the final peptide and under reaction conditions thatwill not alter the target peptide.

[0051] The side-chain protecting groups for Tyr includetetrahydropyranyl, tert-butyl, trityl, benzyl, Cbz, Z—Br—Cbz, and2,5-dichlorobenzyl. The side-chain protecting groups for Asp includebenzyl, 2,6-dichlorobenzyl, methyl, ethyl, and cyclohexyl. Theside-chain protecting groups for Thr and Ser include acetyl, benzoyl,trityl, tetrahydropyranyl, benzyl, 2,6-dichlorobenzyl, and Cbz. Theside-chain protecting group for Thr and Ser is benzyl. The side-chainprotecting groups for Arg include nitro, Tosyl (Tos), Cbz,adamantyloxycarbonyl mesitoylsulfonyl (Mts), or Boc. The side-chainprotecting groups for Lys include Cbz, 2-chlorobenzyloxycarbonyl(2Cl—Cbz), 2-bromobenzyloxycarbonyl (2-BrCbz), Tos, or Boc.

[0052] After removal of the alpha-amino protecting group, the remainingprotected amino acids are coupled stepwise in the desired order. Anexcess of each protected amino acid is generally used with anappropriate carboxyl group activator such as dicyclohexylcarbodiimide(DCC) in solution, for example, in methylene chloride (CH₂ Cl₂),dimethyl formamide (DMF) mixtures.

[0053] After the desired amino acid sequence has been completed, thedesired peptide is decoupled from the resin support by treatment with areagent such as trifluoroacetic acid or hydrogen fluoride (HF), whichnot only cleaves the peptide from the resin, but also cleaves allremaining side chain protecting groups. When the chloromethylated resinis used, hydrogen fluoride treatment results in the formation of thefree peptide acids. When the benzhydrylamine resin is used, hydrogenfluoride treatment results directly in the free peptide amide.Alternatively, when the chloromethylated resin is employed, the sidechain protected peptide can be decoupled by treatment of the peptideresin with ammonia to give the desired side chain protected amide orwith an alkylamine to give a side chain protected alkylamide ordialkylamide. Side chain protection is then removed in the usual fashionby treatment with hydrogen fluoride to give the free amides,alkylamides, or dialkylamides.

[0054] These solid phase peptide synthesis procedures are well known inthe art and further described by Stewart and Young, Solid Phase PeptideSyntheses (2nd Ed., Pierce Chemical Company, 1984).

[0055] B. Synthetic Amino Acids

[0056] These procedures can also be used to synthesize peptides in whichamino acids other than the 20 naturally occurring, genetically encodedamino acids are substituted at one, two, or more positions of any of thecompounds describedherein. For instance, naphthylalanine can besubstituted for tryptophan, facilitating synthesis. Other syntheticamino acids that can be substituted into the peptides includeL-hydroxypropyl, L-3, 4-dihydroxy-phenylalanyl, amino acids such asL-d-hydroxylysyl and D-d-methylalanyl, L-α-methylalanyl, β amino acids,and isoquinolyl. D amino acids and non-naturally occurring syntheticamino acids can also be incorporated into the peptides.

[0057] One can replace the naturally occurring side chains of the 20genetically encoded amino acids (or D amino acids) with other sidechains, for instance with groups such as alkyl, lower alkyl, cyclic 4-,5-, 6-, to 7-membered alkyl, amide, amide lower alkyl, amide di(loweralkyl), lower alkoxy, hydroxy, carboxy and the lower ester derivativesthereof, and with 4-, 5-, 6-, to 7-membered hetereocyclic. Inparticular, proline analogs in which the ring size of the prolineresidue is changed from 5 members to 4, 6, or 7 members can be employed.Cyclic groups can be saturated or unsaturated, and if unsaturated, canbe aromatic or non-aromatic. Heterocyclic groups preferably contain oneor more nitrogen, oxygen, and/or sulphur heteroatoms. Examples of suchgroups include the furazanyl, furyl, imidazolidinyl, imidazolyl,imidazolinyl, isothiazolyl, isoxazolyl, morpholinyl (e.g. morpholino),oxazolyl, piperazinyl (e.g., 1-piperazinyl), piperidyl (e.g.,1-piperidyl, piperidino), pyranyl, pyrazinyl, pyrazolidinyl,pyrazolinyl, pyrazolyl, pyridazinyl, pyridyl, pyrimidinyl, pyrrolidinyl(e.g., 1-pyrrolidinyl), pyrrolinyl, pyrrolyl, thiadiazolyl, thiazolyl,thienyl, thiomorpholinyl (e.g., thiomorpholino), and triazolyl. Theseheterocyclic groups can be substituted or unsubstituted. Where a groupis substituted, the substituent can be alkyl, alkoxy, halogen, oxygen,or substituted or unsubstituted phenyl.

[0058] One can also readily modify the peptides by phosphorylation (see,e.g., Bannwarth et al., 1996, Biorganic and Medicinal Chemistry Letters,6(17):2141-2146), and other methods for making peptide derivatives ofthe compounds disclosed herein are described in Hruby et al., 1990,Biochem J 268:249-262. Thus, the peptide compounds can also serve as abasis to prepare peptide mimetics with similar biological activity.

[0059] C. Terminal Modifications

[0060] Those of skill in the art recognize that a variety of techniquesare available for constructing peptide mimetics with the same or similardesired biological activity as the corresponding peptide compound butwith more favorable activity than the peptide with respect tosolubility, stability, and susceptibility to hydrolysis and proteolysis.See, e.g., Morgan et al., 1989, Ann Rep Med Chem 24:243-252. Thefollowing describes methods for preparing peptide mimetics modified atthe N-terminal amino group, the C-terminal carboxyl group, and/orchanging one or more of the amido linkages in the peptide to a non-amidolinkage. It being understood that two or more such modifications can becoupled in one peptide mimetic structure (e.g., modification at theC-terminal carboxyl group and inclusion of a —CH₂ -carbamate linkagebetween two amino acids in the peptide).

[0061] 1. N-terminal Modifications

[0062] The peptides typically are synthesized as the free acid but, asnoted above, could be readily prepared as the amide or ester. One canalso modify the amino and/or carboxy terminus of the peptide compoundsto produce other compounds . Amino terminus modifications includemethylation (i.e., —NHCH₃ or —NH(CH₃)₂), acetylation, adding abenzyloxycarbonyl group, or blocking the amino terminus with anyblocking group containing a carboxylate functionality defined by RCOO—,where R is selected from the group consisting of naphthyl, acridinyl,steroidyl, and similar groups. Carboxy terminus modifications includereplacing the free acid with a carboxamide group or forming a cycliclactam at the carboxy terminus to introduce structural constraints.

[0063] Amino terminus modifications are as recited above and includealkylating, acetylating, adding a carbobenzoyl group, forming asuccinimide group, etc. (see, e.g., Murray et al., Burger's MedicinalChemistry and Drug Discovery, 5th Ed., Vol. 1, Wolf, ed., John Wiley andSons, Inc. (1995)) Specifically, the N-terminal amino group can then bereacted as follows:

[0064] a) to form an amide group of the formula RC(O)NH— where R is asdefined above by reaction with an acid halide (e.g., RC(O)Cl) orsymmetric anhydride. Typically, the reaction can be conducted bycontacting about equimolar or excess amounts (e.g., about 5 equivalents)of an acid halide to the peptide in an inert diluent (e.g.,dichloromethane) preferably containing an excess (e.g., about 10equivalents) of a tertiary amine, such as diisopropylethylamine, toscavenge the acid generated during reaction. Reaction conditions areotherwise conventional (e.g., room temperature for 30 minutes).Alkylation of the terminal amino to provide for a lower alkylN-substitution followed by reaction with an acid halide as describedabove will provide for N-alkyl amide group of the formula RC(O)NR—; or

[0065] b) to form a succinimide group by reaction with succinicanhydride. As before, an approximately equirnolar amount or an excess ofsuccinic anhydride (e.g., about 5 equivalents) can be employed and theamino group is converted to the succinimide by methods well known in theart including the use of an excess (e.g., ten equivalents) of a tertiaryamine such as diisopropylethylamine in a suitable inert solvent (e.g.,dichloromethane). See, for example, Wollenberg et al., U.S. Pat. No.4,612,132 which is incorporated herein by reference in its entirety. Itis understood that the succinic group can be substituted with, forexample, C₂-C₆ alkyl or —SR substituents which are prepared in aconventional manner to provide for substituted succinimide at theN-terminus of the peptide. Such alkyl substituents are prepared byreaction of a lower olefin (C₂-C) with maleic anhydride in the mannerdescribed by Wollenberg et al., supra and —SR substituents are preparedby reaction of RSH with maleic anhydride where R is as defined above; or

[0066] c) to form a benzyloxycarbonyl-NH— or a substitutedbenzyloxycarbonyl-NH— group by reaction with approximately an equivalentamount or an excess of CBZ—Cl (i.e., benzyloxycarbonyl chloride) or asubstituted CBZ—Cl in a suitable inert diluent (e.g., dichloromethane)preferably containing a tertiary amine to scavenge the acid generatedduring the reaction; or

[0067] d) to form a sulfonamide group by reaction with an equivalentamount or an excess (e.g., 5 equivalents) of R—S(O)₂ Cl in a suitableinert diluent (dichloromethane) to convert the terminal amine into asulfonamide where R is as defined above. Preferably, the inert diluentcontains excess tertiary amine (e.g., ten equivalents) such asdiisopropylethylamine, to scavenge the acid generated during reaction.Reaction conditions are otherwise conventional (e.g., room temperaturefor 30 minutes); or

[0068] e) to form a carbamate group by reaction with an equivalentamount or an excess (e.g., 5 equivalents) of R—OC(O)Cl or R—OC(O)OC₆ H₄-p-NO₂ in a suitable inert diluent (e.g., dichloromethane) to convertthe terminal amine into a carbamate where R is as defined above.Preferably, the inert diluent contains an excess (e.g., about 10equivalents) of a tertiary amine, such as diisopropylethylamine, toscavenge any acid generated during reaction. Reaction conditions areotherwise conventional (e.g., room temperature for 30 minutes); or

[0069] f) to form a urea group by reaction with an equivalent amount oran excess (e.g., 5 equivalents) of R—N═C═O in a suitable inert diluent(e.g., dichloromethane) to convert the terminal amine into a urea (i.e.,RNHC(O)NH—) group where R is as defined above. Preferably, the inertdiluent contains an excess (e.g., about 10 equivalents) of a tertiaryamine, such as diisopropylethylamine. Reaction conditions are otherwiseconventional (e.g., room temperature for about 30 minutes).

[0070] 2. C-terminal Modifications

[0071] In preparing peptide mimetics wherein the C-terminal carboxylgroup is replaced by an ester (i.e., —C(O)OR where R is as definedabove), the resins used to prepare the peptide acids are employed, andthe side chain protected peptide is cleaved with base and theappropriate alcohol, e.g., methanol. Side chain protecting groups arethen removed in the usual fashion by treatment with hydrogen fluoride toobtain the desired ester.

[0072] In preparing peptide mimetics wherein the C-terminal carboxylgroup is replaced by the amide —C(O)NR³ R⁴, a benzhydrylamine resin isused as the solid support for peptide synthesis. Upon completion of thesynthesis, hydrogen fluoride treatment to release the peptide from thesupport results directly in the free peptide amide (i.e., the C-terminusis —C(O)NH₂). Alternatively, use of the chloromethylated resin duringpeptide synthesis coupled with reaction with ammonia to cleave the sidechain protected peptide from the support yields the free peptide amideand reaction with an alkylamine or a dialkylamine yields a side chainprotected alkylamide or dialkylamide (i.e., the C-terminus is —C(O)NRR¹where R and R¹ are as defined above). Side chain protection is thenremoved in the usual fashion by treatment with hydrogen fluoride to givethe free amides, alkylamides, or dialkylamides.

[0073] In another alternative embodiment, the C-terminal carboxyl groupor a C-terminal ester can be induced to cyclize by internal displacementof the —OH or the ester (—OR) of the carboxyl group or esterrespectively with the N-terminal amino group to form a cyclic peptide.For example, after synthesis and cleavage to give the peptide acid, thefree acid is converted to an activated ester by an appropriate carboxylgroup activator such as dicyclohexylcarbodiimide (DCC) in solution, forexample, in methylene chloride (CH₂ Cl₂), dimethyl formamide (DMF)mixtures. The cyclic peptide is then formed by internal displacement ofthe activated ester with the N-terminal amine. Internal cyclization asopposed to polymerization can be enhanced by use of very dilutesolutions. Such methods are well known in the art.

[0074] One can also cyclize the peptides herein, or incorporate adesamino or descarboxy residue at the termini of the peptide, so thatthere is no terminal amino or carboxyl group, to decrease susceptibilityto proteases or to restrict the conformation of the peptide. C-terminalfunctional groups of the compounds include amide, amide lower alkyl,amide di(lower alkyl), lower alkoxy, hydroxy, and carboxy, and the lowerester derivatives thereof, and the pharmaceutically acceptable saltsthereof.

[0075] In addition to the foregoing N-terminal and C-terminalmodifications, the peptide compounds , including peptidomimetics, canadvantageously be modified with or covalently coupled to one or more ofa variety of hydrophilic polymers. It has been found that when thepeptide compounds are derivatized with a hydrophilic polymer, theirsolubility and circulation half-lives are increased and theirimmunogenicity is masked. Quite surprisingly, the foregoing can beaccomplished with little, if any, diminishment in their bindingactivity. Nonproteinaceous polymers suitable for use include, but arenot limited to, polyalkylethers as exemplified by polyethylene glycoland polypropylene glycol, polylactic acid, polyglycolic acid,polyoxyalkenes, polyvinylalcohol, polyvinylpyrrolidone, cellulose andcellulose derivatives, dextran and dextran derivatives, etc. Generally,such hydrophilic polymers have an average molecular weight ranging fromabout 500 to about 100,000 daltons, more preferably from about 2,000 toabout 40,000 daltons and, even more preferably, from about 5,000 toabout 20,000 daltons. In preferred embodiments, such hydrophilicpolymers have an average molecular weights of about 5,000 daltons,10,000 daltons and 20,000 daltons.

[0076] The peptide compounds can be derivatized with or coupled to suchpolymers using, but not limited to, any of the methods set forth inZallipsky, 1995, Bioconjugate Chem 6:150-165 and Monfardini et al.,1995, Bioconjugate Chem 6:62-69, all of which are incorporated byreference in their entirety herein.

[0077] In a presently preferred embodiment, the peptide compounds arederivatized with polyethylene glycol (PEG). PEG is a linear,water-soluble polymer of ethylene oxide repeating units with twoterminal hydroxyl groups. PEGs are classified by their molecular weightswhich typically range from about 500 daltons to about 40,000 daltons. Ina presently preferred embodiment, the PEGs employed have molecularweights ranging from 5,000 daltons to about 20,000 daltons. PEGs coupledto the peptide compounds can be either branched or unbranched. (see,e.g., Monfardini et al., 1995, Bioconjugate Chem 6:62-69). PEGs arecommercially available from Shearwater Polymers, Inc. (Huntsville,Ala.), Sigma Chemical Co. and other companies. Such PEGs include, butare not limited to, monomethoxypolyethylene glycol (MePEG-OH),monomethoxypolyethylene glycol-succinate (MePEG-S),monomethoxypolyethylene glycol-succinimidyl succinate (MePEG-S-NHS),monomethoxypolyethylene glycol-amine (MePEG-NH₂),monomethoxypolyethylene glycol-tresylate (MePEG-TRES), andmonomethoxypolyethylene glycol-imidazolyl-carbonyl (MePEG-IM).

[0078] Briefly, in one exemplar embodiment, the hydrophilic polymerwhich is employed, e.g., PEG, is preferably capped at one end by anunreactive group such as a methoxy or ethoxy group. Thereafter, thepolymer is activated at the other end by reaction with a suitableactivating agent, such as cyanuric halides (e.g., cyanuric chloride,bromide or fluoride), diimadozle, an anhydride reagent (e.g., adihalosuccinic anhydride, such as dibromosuccinic anhydride), acylazide, p-diazoiumbenzyl ether,3-(p-diazoniumphenoxy)-2-hydroxypropylether) and the like. The activatedpolymer is then reacted with a peptide compound disclosed or taughtherein to produce a peptide compound derivatized with a polymer.Alternatively, a functional group in the peptide compounds can beactivated for reaction with the polymer, or the two groups can be joinedin a concerted coupling reaction using known coupling methods. It willbe readily appreciated that the peptide compounds can be derivatizedwith PEG using a myriad of reaction schemes known to and used by thoseof skill in the art.

[0079] In addition to derivatizing the peptide compounds with ahydrophilic polymer (e.g., PEG), it has been discovered that other smallpeptides, e.g., other peptides or ligands that bind to a receptor, canalso be derivatized with such hydrophilic polymers with little, if any,loss in biological activity (e.g., binding activity, agonist activity,antagonist activity, etc.). It has been found that when these smallpeptides are derivatized with a hydrophlilic polymer, their solubilityand circulation half-lives are increased and their immunogenicity isdecreased. Again, quite surprisingly, the foregoing can be accomplishedwith little, if any, loss in biological activity. In fat, in preferredembodiments, the derivatized peptides have an activity that is 0.1 to0.01-fold that of the unmodified peptides. In more preferredembodiments, the derivatized peptides have an activity that is 0.1 to1-fold that of the unmodified peptides. In even more preferredembodiments, the derivatized peptides have an activity that is greaterthan the unmodified peptides.

[0080] Peptides suitable for use in this embodiment generally includethose peptides, i.e., ligands, that bind to members of the Bcl-2receptor family. Such peptides typically comprise about 150 amino acidresidues or less and, more preferably, about 100 amino acid residues orless (e.g., about 10-12 kDa). Hydrophilic polymers suitable foruseherein include, but are not limited to, polyalkylethers asexemplified by polyethylene glycol and polypropylene glycol, polylacticacid, polyglycolic acid, polyoxyalkenes, polyvinylalcohol,polyvinylpyrrolidone, cellulose and cellulose derivatives, dextran anddextran derivatives, etc. Generally, such hydrophilic polymers have anaverage molecular weight ranging from about 500 to about 100,000daltons, more preferably from about 2,000 to about 40,000 daltons and,even more preferably, from about 5,000 to about 20,000 daltons. Inpreferred embodiments, such hydrophilic polymers have an averagemolecular weights of about 5,000 daltons, 10,000 daltons and 20,000daltons. The peptide compounds can be derivatized with using the methodsdescribed above and in the cited references.

[0081] D. Backbone Modifications

[0082] Other methods for making peptide derivatives of the compoundsdescribed herein are described in Hruby et al., 1990, Biochem J268(2):249-262, incorporated herein by reference. Thus, the peptidecompounds also serve as structural models for non-peptidic compoundswith similar biological activity. Those of skill in the art recognizethat a variety of techniques are available for constructing compoundswith the same or similar desired biological activity as the lead peptidecompound but with more favorable activity than the lead with respect tosolubility, stability, and susceptibility to hydrolysis and proteolysis.See Morgan et al., 1989, Ann Rep Med Chem 24:243-252, incorporatedherein by reference. These techniques include replacing the peptidebackbone with a backbone composed of phosphonates, amidates, carbamates,sulfonamides, secondary amines, and N-methylamino acids.

[0083] Peptide mimetics wherein one or more of the peptidyl linkages[—C(O)NH—] have been replaced by such linkages as a —CH₂-carbamatelinkage, a phosphonate linkage, a —CH₂-sulfonamide linkage, a urealinkage, a secondary amine (—CH₂ NH—) linkage, and an alkylated peptidyllinkage [—C(O)NR⁶— where R⁶ is lower alkyl] are prepared duringconventional peptide synthesis by merely substituting a suitablyprotected amino acid analogue for the amino acid reagent at theappropriate point during synthesis.

[0084] Suitable reagents include, for example, amino acid analogueswherein the carboxyl group of the amino acid has been replaced with amoiety suitable for forming one of the above linkages. For example, ifone desires to replace a —C(O)NR— linkage in the peptide with a—CH₂-carbamate linkage (—CH₂ OC(O)NR—), then the carboxyl (—COOH) groupof a suitably protected amino acid is first reduced to the —CH₂ OH groupwhich is then converted by conventional methods to a —OC(O)Clfunctionality or a para-nitrocarbonate —OC(O)O—C₆ H₄ -p-NO₂functionality. Reaction of either of such functional groups with thefree amine or an alkylated amine on the N-terminus of the partiallyfabricated peptide found on the solid support leads to the formation ofa —CH₂ OC(O)NR— linkage. For a more detailed description of theformation of such —CH₂-carbamate linkages, see Cho et al., 1993, Science261:1303-1305.

[0085] Similarly, replacement of an amido linkage in the peptide with aphosphonate linkage can be achieved in the manner set forth in U.S.patent applications Ser. Nos. 07/943,805, 08/081,577 and 08/119,700, thedisclosures of which are incorporated herein by reference in theirentirety.

[0086] Replacement of an amido linkage in the peptide with a—CH₂-sulfonamide linkage can be achieved by reducing the carboxyl(—COOH) group of a suitably protected amino acid to the —CH₂ OH groupand the hydroxyl group is then converted to a suitable leaving groupsuch as a tosyl group by conventional methods. Reaction of the tosylatedderivative with, for example, thioacetic acid followed by hydrolysis andoxidative chlorination will provide for the —CH₂ —S(O)₂ Cl functionalgroup which replaces the carboxyl group of the otherwise suitablyprotected amino acid. Use of this suitably protected amino acid analoguein peptide synthesis provides for inclusion of an —CH₂ S(O)₂ NR— linkagewhich replaces the amido linkage in the peptide thereby providing apeptide mimetic. For a more complete description on the conversion ofthe carboxyl group of the amino acid to a —CH₂ S(O)₂ Cl group, see, forexample, Weinstein, Chemistry & Biochemistry of Amino Acids, Peptidesand Proteins, Vol. 7, pp. 267-357, Marcel Dekker, Inc., New York (1983)which is incorporated herein by reference.

[0087] Replacement of an amido linkage in the peptide with a urealinkage can be achieved in the manner set forth in U.S. patentapplication Ser. No. 08/147,805, which is incorporated herein byreference.

[0088] Secondary amine linkages wherein a CH₂NH linkage replaces theamido linkage in the peptide can be prepared by employing, for example,a suitably protected dipeptide analogue wherein the carbonyl bond of theamido linkage has been reduced to a CH₂ group by conventional methods.For example, in the case of diglycine, reduction of the amide to theamine will yield after deprotection H₂ NCH₂ CH₂ NHCH₂ COOH which is thenused in N-protected form in the next coupling reaction. The preparationof such analogues by reduction of the carbonyl group of the amidolinkage in the dipeptide is well known in the art (see, e.g., Remington,1994, Meth Mol Bio 35:241-247).

[0089] The suitably protected amino acid analogue is employed in theconventional peptide synthesis in the same manner as would thecorresponding amino acid. For example, typically about 3 equivalents ofthe protected amino acid analogue are employed in this reaction. Aninert organic diluent such as methylene chloride or DMF is employed and,when an acid is generated as a reaction by-product, the reaction solventwill typically contain an excess amount of a tertiary amine to scavengethe acid generated during the reaction. One particularly preferredtertiary amine is diisopropylethylamine which is typically employed inabout 10 fold excess. The reaction results in incorporation into thepeptide mimetic of an amino acid analogue having a non-peptidyl linkage.Such substitution can be repeated as desired such that from zero to allof the amido bonds in the peptide have been replaced by non-amido bonds.

[0090] One can also cyclize the peptides described herein, orincorporate a desamino or descarboxy residue at the terminii of thepeptide, so that there is no terminal amino or carboxyl group, todecrease susceptibility to proteases or to restrict the conformation ofthe peptide. C-terminal functional groups of the compounds includeamide, amide lower alkyl, amide di(lower alkyl), lower alkoxy, hydroxy,and carboxy, and the lower ester derivatives thereof, and thepharmaceutically acceptable salts thereof.

[0091] E. Disulfide Bond Formation

[0092] The compounds described herein may exist in a cyclized form withan intramolecular disulfide bond between the thiol groups of thecysteines. Alternatively, an intermolecular disulfide bond between thethiol groups of the cysteines can be produced to yield a dimeric (orhigher oligomeric) compound. One or more of the cysteine residues mayalso be substituted with a homocysteine.

[0093] Other embodiments of this invention provide for analogs of thesedisulfide derivatives in which one of the sulfurs has been replaced by aCH₂ group or other isostere for sulfur. These analogs can be made via anintramolecular or intermolecular displacement, using methods known inthe art.

[0094] Alternatively, the amino-terminus of the peptide can be cappedwith an alpha-substituted acetic acid, wherein the alpha substituent isa leaving group, such as an α-haloacetic acid, for example,α-chloroacetic acid, α-bromoacetic acid, or α-iodoacetic acid. Thecompounds can be cyclized or dimerized via displacement of the leavinggroup by the sulfuir of the cysteine or homocysteine residue. See, e.g.,Andreu et al., 1994, Meth Mol Bio 35(7):91-169; Barker et al., 1992, JMed Chem 35:2040-2048; and Or et al., 1991, J Org Chem 56:3146-3149,each of which is incorporated herein by reference.

[0095] The present peptides may also be prepared by recombinant DNAtechniques well known in the art.

IV. Antibody Preparation

[0096] Antibodies against the loop domain of Bcl-2. The N-terminal loopregion of Bcl-2 can be expressed and used as an antigen to developanti-Bcl-2/N-terminal loop region antibodies. These antibodies, bybinding to the loop domain of Bcl-2, can mimic the activity of TR3 inthe induction of a conformational change of Bcl-2 which will expose thehidden BH3 domain and confer pro-apoptotic activity to the Bcl-2protein.

[0097] Further, Bcl-X_(L) can be expressed and used as an antigen todevelop anti-Bcl-X_(L) antibodies. Antibodies which mimic the activityof TCTP may be identified. The antibodies may then be used fortherapeutics and diagnostics comparably to those identified for Bcl-2.

[0098] The terms “antibody” or “antibody peptide(s)” refer to an intactantibody, or a binding fragment thereof that competes with the intactantibody for specific binding. Binding fragments are produced byrecombinant DNA techniques, or by enzymatic or chemical cleavage ofintact antibodies. Binding fragments include Fab, Fab′, F(ab′)₂, Fv, andsingle-chain antibodies. An antibody other than a “bispecific” or“bifunctional” antibody is understood to have each of its binding sitesidentical. An antibody substantially inhibits adhesion of a receptor toa counterreceptor when an excess of antibody reduces the quantity ofreceptor bound to counterreceptor by at least about 20%, 40%, 60% or80%, and more usually greater than about 85% (as measured in an in vitrocompetitive binding assay).

[0099] The term “epitope” includes any protein determinant capable ofspecific binding to an immunoglobulin or T-cell receptor. Epitopicdeterminants usually consist of chemically active surface groupings ofmolecules such as amino acids or sugar side chains and usually havespecific three dimensional structural characteristics, as well asspecific charge characteristics. An antibody is said to specificallybind an antigen when the dissociation constant is ≦1 μM, preferably ≦100nM and most preferably ≦10 nM.

Antibody Structure

[0100] The basic antibody structural unit is known to comprise atetramer. Each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kDa) and one“heavy” chain (about 50-70 kDa). The amino-terminal portion of eachchain includes a variable region of about 100 to 110 or more amino acidsprimarily responsible for antigen recognition. The carboxy-terminalportion of each chain defines a constant region primarily responsiblefor effector function. Human light chains are classified as kappa andlambda light chains. Heavy chains are classified as mu, delta, gamma,alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgA,and IgE, respectively. Within light and heavy chains, the variable andconstant regions are joined by a “J” region of about 12 or more aminoacids, with the heavy chain also including a “D” region of about 10 moreamino acids. See generally, Fundamental Immunology Ch. 7 (Paul, W., ed.,2nd ed. Raven Press, N.Y. (1989)) (incorporated by reference in itsentirety for all purposes). The variable regions of each light/heavychain pair form the antibody binding site. Thus, an intact antibody hastwo binding sites. Except in bifunctional or bispecific antibodies, thetwo binding sites are the same.

[0101] The chains all exhibit the same general structure of relativelyconserved framework regions (FR) joined by three hyper variable regions,also called complementarity determining regions or CDRs. The CDRs fromthe two chains of each pair are aligned by the framework regions,enabling binding to a specific epitope. From N-terminal to C-terminal,both light and heavy chains comprise the domains FR1, CDR1, FR2, CDR2,FR3, CDR3 and FR4. The assignment of amino acids to each domain is inaccordance with the definitions of Kabat Sequences of Proteins ofImmunological Interest (National Institutes of Health, Bethesda, Md.(1987 and 1991)), or Chothia & Lesk J. Mol. Biol. 196:901-917 (1987);Chothia et aL Nature 342:878-883 (1989).

[0102] A bispecific or bifunctional antibody is an artificial hybridantibody having two different heavy/light chain pairs and two differentbinding sites. Bispecific antibodies can be produced by a variety ofmethods including fusion of hybridomas or linking of Fab′ fragments.See, e.g., Songsivilai & Lachmann Clin. Exp. Immunol. 79: 315-321(1990), Kostelny et al. J. Immunol. 148:1547-1553 (1992). Production ofbispecific antibodies can be a relatively labor intensive processcompared with production of conventional antibodies and yields anddegree of purity are generally lower for bispecific antibodies.Bispecific antibodies do not exist in the form of fragments having asingle binding site (e.g., Fab, Fab′, and Fv).

[0103] Human Antibodies and Humanization of Antibodies—Human antibodiesavoid certain of the problems associated with antibodies that possessmurine or rat variable and/or constant regions. The presence of suchmurine or rat derived proteins can lead to the rapid clearance of theantibodies or can lead to the generation of an immune response againstthe antibody by a patient. In order to avoid the utilization of murineor rat derived antibodies, fully human antibodies can be generated usingmethods well known to those of skill in the art.

[0104] Therapeutic Administration and Formulations—It will beappreciated that therapeutic entities will be administered with suitablecarriers, excipients, and other agents that are incorporated intoformulations to provide improved transfer, delivery, tolerance, and thelike. A multitude of appropriate formulations can be found in theformulary known to all pharmaceutical chemists: Remington'sPharmaceutical Sciences (18^(th) ed, Mack Publishing Company, Easton,Pa. (1990)), particularly Chapter 87 by Block, Lawrence, therein. Theseformulations include, for example, powders, pastes, ointments, jellies,waxes, oils, lipids, lipid (cationic or anionic) containing vesicles(such as Lipofectin™), DNA conjugates, anhydrous absorption pastes,oil-in-water and water-in-oil emulsions, emulsions carbowax(polyethylene glycols of various molecular weights), semi-solid gels,and semi-solid mixtures containing carbowax. Any of the foregoingmixtures can be appropriate in treatments and therapies, provided thatthe active ingredient in the formulation is not inactivated by theformulation and the formulation is physiologically compatible andtolerable with the route of administration. See also Baldrick P.“Pharmaceutical excipient development: the need for preclinicalguidance.” Regul. Toxicol. Pharmacol. 32(2):210-8 (2000), Wang W.“Lyophilization and development of solid protein pharmaceuticals.” Int.J. Pharm. 203(1-2):1-60 (2000), Charman W N “Lipids, lipophilic drugs,and oral drug delivery-some emerging concepts.” J Pharm Sci0.89(8):967-78 (2000), Powell et al. “Compendium of excipients forparenteral formulations” PDA J Pharm Sci Technol. 52:238-311 (1998) andthe citations therein for additional information related toformulations, excipients and carriers well known to pharmaceuticalchemists.

[0105] For diagnostic uses, any type of label (detectable moiety) may beadded to the antibody, including but not limited to, for example, aradiolabel, flourescent label, enzymatic label chemiluminescent lable ora biotinyl group. Radioisotopes or radionuclides can include ³H, ¹⁴C,¹⁵N, ³⁵S, ⁹⁰Y, ⁹⁹Tc, ¹¹¹In, ¹²⁵I, ¹³¹I, fluorescent lables can includerhodamine, lanthanide phosphors or FITC and enzymatic labels can includehorseradish peroxidase, β-galactosidase, luciferase, alkalinephosphatase.

V. Intrabody Preparation

[0106] Intrabodies are scFvs that are expressed within the cell anddirected against intracellular proteins. In this way they can interfereand inhibit cellular processes inside the cell in a number of ways.Intrabodies can inhibit an enzymatic activity directly, or interferewith protein-protein interactions, thus disrupting signaling pathways.They can also be used to displace a protein from its site of action. Thefusion of intracellular localization signals, such as a nuclearlocalization signal (NLS) or a retention signal for the endoplasmicreticulum (ER), can be used to re-direct the antibody and its targetantigen to specific locations within the cell. For instance, an scFvdirected against the ErbB-2 receptor and designed to prevent transitthrough the ER was shown to down-regulate the surface expression ofErbB-2 and consequently, to considerably affect growth factor signaling.

[0107] Thus, it is envisioned that intrabodies can be used to mimic theaction of TR3 in a cell to cause apoptosis. Alternatively, intrabodiescan be used to interfere with TR3 binding to Bcl-2 to keep cells in aproliferative state. Intrabodies could be advantageously used for thetreatment of leukemias and lymphomas because it can be envisioned thatthe involved blood cells could be more easily removed from a patient,genetically manipulated to include the intrabody, and introduced backinto the body of the patient.

[0108] Methods of selection of intrabodies includes the use of phagedisplay libraries, ribosome and mRNA display, protein fragmentcomplementation assay (PCA), and yeast screening assays which are anadaptation of the two hybrid system using scFvs and an antigen.

EXAMPLES

[0109] The following examples describe the processes used to identifyand characterize peptides that target Bcl-2-family members and regulatetheir apoptotic functions. Unless otherwise indicated, the plasmids andmethods were as follows:

[0110] Plasmids—Plasmids encoding TR3 and TR3/ΔDBD (Li, et al. 2000,Science 289, 1159-1164), Bcl-2, Bcl-Gs, L216E-Bcl-Gs, Bcl-2/Δloop (Chenget aL 1997, Science 278, 1966-1968), Bax, and Bcl-X_(L) (Guo, et al.2001, J. Biol. Chem. 276, 2780-2785), have been described previously. Toconstruct N168, DC3, DC1, TR3/ΔDBD/DC1, TR3/ΔDBD/Δ471-488, Bcl-2/1-80,appropriate TR3 or Bcl-2 fragments were prepared either by restrictionenzyme digestion or amplified by polymerase chain reaction (PCR). Theresulting TR3 fragments were then cloned into pGFP-N2 vector (Clontech,USA). TR3/ΔDBD/L487A were cloned by substituting Leu487 with Ala by PCRsite-directed mutagenesis on the TR3/ΔDBD template. Bcl-2/Y108K,Bcl-2/L137A, Bcl-2/G145A, and Bcl-2/R146 were constructed bysubstituting Tyr108, Leu137, Gly145 and Arg146 with Lys, Ala, Ala, andGlu, respectively, by PCR site-directed mutagenesis using the Bcl-2 cDNAas a template. Bcl2/ΔBH1, Bcl-2/ΔBH2, Bcl-2/ΔBH3, Bcl-2/ΔBH4, Bcl-2/ΔTMare deletions of 132-160, 189-204, 90-114, 7-30, 205-239 amino acids inBcl-2 (Hanada et al. 1995 J. Biol. Chem. 270, 11962-11969). Allmutations were confirmed by DNA sequencing.

[0111] Bcl-2 siRNAs and antisense oligonucleotides—The target siRNASMARTpools for Bcl-2 and Bak and the siRNA oligonucleotide for TR3(5′-CAG UCC AGC CAU GCU CCU dTdT) (SEQ ID NO:2) were purchased fromDharmacon Research Inc. Target or control siRNA were transfected at afinal concentration of 200 nM into cells at 40% confluency usingOligofectamine reagent (Invitrogen) according to the manufacturer'srecommendations. After 48 h, cells were analyzed. Bcl-2 antisenseoligonucleotide targeting Bcl-2 and negative control oligonucleotideswere obtained from Calbiochem. They (2.5 μM) were transfected into cellsat 60% confluency for 36 h before analysis.

[0112] TR3/Bcl-2 interaction assays—Reporter gene assays usingNurRE-tk-CAT in CV-1 cells, and GST pull-down assay were describedpreviously (Li, et al. 2000 Science 289, 1159-1164). For the mammaliantwo-hybrid assays, CV-1 cells were co-transfected with pcDNA-Gal4TAD-TR3or pcDNA-Gal4TAD-TR3/ΔDBD and pcDNA-Gal4DBD-Bcl-2/ΔTM along with aluciferase reporter gene driven by four copies of the Gal4-binding site.The cells were harvested and reporter gene activity was measured. ForCo-IP assays, HEK293T cells were transiently transfected with variousexpression plasmids using a modified calcium phosphate precipitationmethod (Wu et al, 1997) in the presence of caspase inhibitors (zVAD-fmk)to prevent degradation of TR3 protein due to apoptosis. Cells weresuspended in lysis buffer (50 mM Tris-HCI, PH7.4; 150 mM NaCl; 20 mMEDTA; 1% NP-40; 1 mM PMSF; 50 μg/ml Leupeptin; 20 mg/ml Aprotinin; 0.1mM Na₃ VO₄; and 1 mM DTT). Cells extracts were cleared by incubationwith the Protein A/G plus Agarose beads (Santa Cruz) and then incubatedwith appropriate antibody and 30 μl of Protein A or G plus Agarose beadsovernight at 4° C. Beads were then washed and boiled in Laemmligel-loading solution before performing SDS-PAGE/immunoblotting using thefollowing polyclonal or monoclonal antibodies: monoclonal mouse anti-GFP(Medical and Biological Laboratories), monoclonal mouse anti-HA (RocheMolecular Biochemicals), monoclonal mouse anti-FLAG (Sigma), monoclonalmouse anti-Myc (Santa Cruz), polyclonal rabbit anti-TR3 (Active Motif),or monoclonal mouse anti-Bcl-2 (Santa Cruz). Immunoreactive productswere detected by chemiluminescence with an enhanced chemiluminescencesystem (ECL) (Amersham).

[0113] Subcellular localization assays—Cells were seeded ontocover-slips in 6-well plates overnight, then transiently transfectedwith GFP-fusion expression plasmids. After 16 hours, cells were washedwith PBS and fixed in 4% paraformaldehyde. For mitochondrial staining,cells were then incubated with anti-Hsp60goat IgG (Santa Cruz, USA),followed by anti-goat IgG conjugated with Cy3 (Sigma). For cyt cstaining, cells were incubated with monoclonal anti-cyt c IgG(PharMingen), followed by anti-mouse IgG conjugated with Cy5 (Amersham).Fluorescent images were collected and analyzed using a MRC-1024 MPlaser-scanning confocal microscope (Bio-Rad). Subcellular fractionationassays were performed as described (Li, et al. 2000 Science 289,1159-1164). Briefly, cells (1×10⁷ cells) suspended in 0.5 ml hypotonicbuffer (5 mM Tris-HCl, pH 7.4, 5 mM KCl, 1.5 mM MgCl₂, 0.1 mM EGTA, pH8.0, and 1 mM DTT) were homogenized and cell extracts were centrifugedat 500×g for 5 min. The resulting supernatant was centrifuged at10,000×g for 30 min at 4° C. to obtain the HM fraction. HM fraction wasresuspended in 100 μl lysis buffer (10 mM Tris, pH 7.4, 150 mM NaCl, 1%Triton X-100, 5 mM EDTA, pH 8.0) for immunoblotting analysis.

[0114] Apoptosis assays—For nuclear morphological change analysis, cellswere trypsinized, washed with PBS, fixed with 3.7% paraformaldehyde, andstained with DAPI (4,6-diamidino-2-phenylindole) (50 μg/ml) to visualizethe nuclei by UV-microscopy. The percentages of apoptotic cells weredetermined by counting 300 GFP-positive cells, scoring cells havingnuclear fragmentation and/or chromatin condensation.

[0115] Isolation and transfection of human peripheral blood lymphocytes(PBLs)—Leukocyte-enriched buffy coats from San Diego Blood Bank werediluted with 2 volumes of PBS, and PBLs were isolated by centrifuge onFicoll-paque™ Plus (Amersham Pharmacia biotech). The mononuclear cellswere cultured in RPMI containing 10% FBS and 20 μM Hepes. Freshlyisolated cells (10⁷ cells per transfection) were washed once with PBScontaining 0.5% BSA and supernatant was completely discarded so that noresidual PBS/BSA covers the pellet. The cells were resuspended in 100 μlof the human T Cell Nucleofector™ solution (Amexa biosystems) and verygently mixed with 1-4 μg DNA in 5 μl. The cell suspension wastransferred into a cuvette supplied by Amexa biosystems andelectroporated using U-14 program of Nucleofector™ device. The cellswere removed from the cuvette quickly by adding 500 μl of the pre-warmedRPMI medium containing 10% FBS and 20 μM Hepes and transferred into 12well plates containing 1.5 μl of pre-warmed culture medium.

Example 1 AHPN, TR3 and Apoptosis in Prostate, Lung and Breast CancerCells

[0116] Retinoid AHPN(6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalenecarboxylic acid)potently induces apoptosis of prostate cancer cells by inducingtranslocation of TR3 from the nucleus to mitochondria (Li et al., 2000,Science 289:1159-1164). AHPN and its analogs also effectively induceapoptosis of lung and breast cancer cells (Li et al., 1998, Mol Cell Bio18:4719-4731; Shao et al., 1995, Oncogene, 11:493-504). TR3 expressionwas significantly induced by AHPN in lung cancer cells. Moreover,inhibition of TR3 expression by overexpressing TR3 antisense RNAabolished apoptosis by AHPN, indicating that TR3 is essential forAHPN-induced apoptosis of lung cancer cells (Li et al., 1998, supra).

[0117] Given the results of these studies, it was desireable todetermine whether TR3 exerts its apoptotic effect in lung cancer cellsas in prostate cancer cells by targeting to mitochondria. GFP-TR3/ΔDBD(a TR3 mutant that lacks the DNA-binding domain) was transientlytransfected into NCI-H460 lung cancer cells. The cells were stained formitochondria (Hsp60) and cytochrome c (Cyt c), and analyzed by confocalmicroscopy. GFP-TR3/ΔDBD expressed in lung cancer cells displaced adistribution pattern overlaid extensively with that of heat shockprotein 60 (HSP60), a mitochondrial specific protein. These dataindicate that GFP-TR3/ΔDBD targeted to mitochondria in lung cancercells, as it did in prostate cancer cells.

[0118] TR3 is also expressed in both estrogen-dependent ZR-75-1 and inestrogen-independent MDA-MB-231 breast cancer cells (Wu et al., 1997,Mol Cell Bio 17:6598-6608). It was previously shown that AHPNeffectively induces apoptosis of both types of breast cancer cell lines.To determine whether TR3 also mediated the apoptotic effect of AHPN andits analogs in breast cancer cells, MDA-MB-231 breast cancer cells weretreated with MM11453, which effectively induces apoptosis ofestrogen-dependent and -independent breast cancer cells. Total RNAs wereprepared from MDA-MB-231 cells treated with MM1153 (10⁻⁶M), MM11384(10⁻⁶M), TPA (100 ng/ml) or EGF (200 ng/ml) for 3 h. and analyzed byNorthern blotting. Upon MM11453 treatment, TR3 expression in MDA-MB-231cells was significantly induced. TR3 expression was also induced byanother apoptosis-inducing retinoid (MM11384), TPA, and the mitogenicagent epidermal growth factor (EGF).

[0119] To study whether the TR3 nuclear-to-mitochondrial targetingpathway also occurs in breast cancer cells, a GFP-TR3 fusion constructwas transfected into MDA-MB-231 cells. GFP-TR3-transfected MDA-MB231cells were treated with or without MM11453 (10⁻⁶M) or MM11384 (10⁻⁶M)for 1 hour, then immunostained with anti-Hsp60antibody (Sigma) followedby Cy3-conjugated secondary antibody (Sigma) to detect mitochondria.GFP-TR3 and mitochondria (Hsp60) were visualized using confocalmicroscopy and the two images were overlaid. The GFP-TR3 fusion proteinwas predominately present in the nucleus in nonstimulated cells.However, upon treatment with MM11453 and MM11384, GFP-TR3 translocatedto mitochondria. Next, GFP-TR3/ΔDBD was transiently transfected intoZR-75-1 or MDA-MB-231 cells, stained for mitochondria (Hsp60) andcytochrome c (Cyt c), and analyzed by confocal microscopy. GFP-TR3/ΔDBDwas targeted to mitochondria of MDA-MB-231 and ZR-75-1 cells in theabsence of any treatment and caused release of cytochrome c from themitochondria.

[0120] Cellular localization of TR3 expressed in mammary tumor derivedfrom transgenic mice bearing polyomavirus middle T antigen, was alsostudied (provided by Dr. W. Muller, see Hutchinson et al., 2001, MolCell Bio 21:2203-2212). The tissue sample was analyzed by the TdT assayfor apoptosis. Mammary tumor tissue from transgenic mice bearingpolyomavirus middle T antigen was stained with TR3 followed byCy3-conjugated secondary antibody to detect TR3 expression andsubcellular localization by confocal microscopy. The tissue was alsoanalyzed by the fluorescein conjugated TdT enzyme (Oncogene) to detectDNA fragmentation (TdT-labeled cells are indicated by green color). Twoimages were overlaid to assess the correlation between apoptosis and TR3subcellular localization. The result showed that TR3 was localized inthe cytoplasm in cells undergoing extensive apoptosis, while it wasfound in the nucleus in nonapoptotic cells. Thus, subcellularlocalization of TR3 plays a role in regulating mammary tumor cellapoptosis.

Example 2 Interaction of TR3 with Bcl-2

[0121] The possibility that TR3 targeted mitochondria by interactingwith Bcl-2 was investigated by first identifying whether the twoproteins interact directly. This was done using a variety of methodsincluding co-immunoprecipitation assays, protein binding assays,two-hybrid assays, co-localization assays and NurRE-dependent reportergene assays. In all cases a direct interaction between TR3 and Bcl-2 wasidentified.

[0122] A. co-immunoprecipitation assays—to identify the interactionbetween TR3 and Bcl-2, a monoclonal antibody was generated against theligand-binding domain (GBD) of TR3 for co-immunoprecipitation (Co-IP)assays. LNCaP cells were treated with TPA to induce endogenous TR3expression for 3 hours. Cell extracts were prepared from TPA-treated andnon-treated cells and incubated with mouse monoclonal anti-TR3 antibody.Immunoblotting of immunoprecipitates employed anti-Bcl-2 antibody orrabbit polyclonal anti-TR3 anti-body (Active Motif). Bcl-2 wasspecifically co-immunoprecipitated by anti-TR3 antibody in TPA-treatedcells, but not in non-treated cells.

[0123] In addition to TPA, AHPN and its structural analogs also induceTR3 expression and TR3-dependent apoptosis in an epithelial cancer cellline. Lysates from H460 lung cancer cells were treated with the AHPNanalog 3-Cl-AHPC (also called MM002) at 10⁻⁶ M for 3 hour, whichpotently induces TR3 expression, mitochondrial targeting and apoptosisin these cells. Lysates were then incubated with anti-Bcl-2 antibody.Immunoblotting of immunoprecipitates was conducted using anti-TR3 andanti-Bcl-2 antibodies. This also demonstrated co-immunoprecipitation ofTR3 with anit-Bcl-2 antibody.

[0124] TR3/ΔDBD, which can constitutively reside on mitochondria , wasalso analyzed by co-immunoprecipitation (Co-IP) assay for itsinteraction with Bcl-2. GFP-TR3/ΔDBD was transfected into humanembryonic kidney cell line 293T alone or together with a Bcl-2expression vector. The expressed GFP-TR3/ΔDBD mutant protein as thenprecipitated by using either anti-Bcl-2 antibody or control IgG anddetected by western blotting using anti-GFP antibody. The same membraneswere also blotted with anti-Bcl-2 antibody to determine precipitationspecificity and efficiency. Input represents 10% of total cell extractused in the precipitation assays. These co-IP assays showed that asignificant amount of TR3/ΔDBD was co-precipitated with Bcl-2 byanti-Bcl-2 antibody .

[0125] B. NurRE-dependent reporter gene assay. To investigate theinhibition of TR3-dependent transactivation by Bcl-2, a reporter geneassay was performed. CV-1 cells were transfected with the NurRE-tk-CAT(Li, et al. Science 289, 1159-1164) and the β-gal expression vector (100ng) with or without TR3 expression vector (25 ng) together and with orwithout Bcl-2 or BAX expression vector. CAT activity was thendetermined. Transactivation of TR3 on its responsive element(NurRE-tk-CAT) was potently inhibited by cotransfection of Bcl-2 but notBAX (FIG. 1), confirming the interaction between Bcl-2 and TR3.

[0126] C. in vitro protein binding assays. To further investigate theinteraction between TR3/ΔDBD and Bcl-2, a GST pull-down assay wasperformed. GST-TR3, GST or GST-RXR immobilized on 20 μl ofglutathione-Sepharose was incubated with 10 μl of in vitro synthesized³⁵S-labeled Bcl-2, RXRα, TR3, or Bax (10 μl). Bound proteins wereanalyzed by SDS-PAGE autoradiography. ³⁵S-labeled Bcl-2 was pulled downby GST-TR3 but not by GST. RXRα, a known heterodimerization partner ofTR3 was also pulled down by GST-TR3. Conversely, ³⁵S-labeled TR3 andBax, a known heterodimerization partner of Bcl-2, bound equally toGST-Bcl-2 but not to GST.

[0127] D. co-localization in cells—First the interaction of endogenousTR3 and transfected Bcl-2 colocalization was identified in cells. Thephorbol ester 12-0-tetradecanoyl phorbol-13-acetate (TPA) induces theexpression of endogenous TR3 and its mitochondrial localization in LNCaPprostate cancer cells. LNCaP cells were transfected with Bcl-2expression vector, treated with or without TPA (100 ng/ml) for 3 h, thenimmunostained with anti-Bcl-2 antibody (Santa Cruz) followed byCy5-conjugated secondary antibody (Amersham Biosciences), or with mousemonoclonal anti-TR3 antibody followed by Cy3-conjugated secondaryantibody (Sigma). Endogenous TR3 and transfected Bcl-2 were visualizedusing confocal microscopy and images were overlaid. To demonstrate themitochondrial localization of TR3 and Bcl-2, cells were also stainedwith Hsp60antibody followed by FITC-conjugated secondary antibody andimages were overlaid. Approximately 80% of TPA-treated cellsdemonstrated colocalization. TR3 was not detected prior to TPAtreatment. However, strong TR3 immunostaining occurred after treatment.In TPA-stimulated cells, the distribution patterns of endogenous TR3 andtransfected Bcl-2 overlapped extensively in the cytoplasm, colocalizingwith Hsp60, a mitochondria-specific protein.

[0128] Next, the interaction of transfected TR3 and Bcl-2 was analyzed.Expression vectors for GFP-TR3/ΔDBD, (GFP-(DBD) and Bcl-2 werecotransfected into LNCaP cells. After 20 h, cells were inimunostainedwith anti-Bcl-2 antibody followed by Cy3-conjugated secondary antibodies(Sigma). GFP-fusion and Bcl-2 were visualized using confocal microscopy,and the two images were overlaid. For a control, the distribution oftransfected GFP empty vector was analyzed. Approximately 30% oftransfected cells exhibited colocalization. These studies demonstratedthat TR3 interacts specifically with Bcl-2.

[0129] E. Two-hybrid assay—a two-hybrid assay was performed to assessthe interaction of TR3 and Bcl-2. The C-terminal trans-membrane domain(TM) was deleted from Bcl-2 to prevent its membrane accumulation. Gal4reporter gene (Gal4)₂-tk-CAT) (250 ng) and β-gal expression plasmids (50ng) were co-transfected into CV-1 cells with the Bcl-2/ΔTM lacking thetrans-membrane domain or RXRα fused with the Gal4 DNA-binding domain(Gal-DBD) alone or with the TR3 or TR3/ΔDBD fused with the Gal4transactivation domain (Gal-TAD). Reporter gene activity was determined48 h later and normalized relative to β-gal activity. Bcl-2/ΔTM fusedwith the Gal4-DNA-binding domain (DBD) strongly activated a reportercontaining a Gal4-response element when co-expressed with the Gal4transactivation-domain (TAD) fused with TR3 or TR3/ΔDBD, a TR3 mutantlacking its DBD. Comparable activation was observed upon co-transfectionof the Gal4-TAD-TR3 fusion with an expression vector containing RXRαfused with Gal4-DBD.

Example 3 The Domain of TR3 which Interacts with Bcl-2

[0130] Several TR3 mutants were analyzed to determine the domain of TR3which interacts with Bcl-2. The TR3 mutants are schematicallyrepresented in FIG. 1. A Co-IP assay was performed, as described above.Briefly, plasmids encoding these TR3 mutant GFP fusions were transfectedinto HEK293T cells, which contained undetectable levels of endogenousBcl-2, with or without a Bcl-2 expression vector (2 μg) (the emptyvector pRC/CMV was used as a control). The empty GFP vector (6 μg) wasalso used as a control. Cell extracts were prepared and incubated withanti-Bcl-2 antibody or control IgG for Co-IP assays. Lysates wereimmunoprecipitated by using either polyclonal rabbit anti-Bcl-2 antibodyor control IgG. Cell lysates and immunoprecipitates were examined byimmunoblotting using anti-GFP antibody. The same membranes were alsoblotted with anti-Bcl-2 antibody (Santa Cruz) to determine IPspecificity and efficiency. When GFP control vector was co-transfectedwith Bcl-2, GFP was not precipitated by anti-Bcl-2 antibody or controlIgG, indicating that GFP did not interact with Bcl-2. However, whenGFP-TR3/ΔDBD was transfected together with the Bcl-2 expression vector,a significant amount of GFP-TR3/ΔDBD was co-precipitated with Bcl-2 byanti-Bcl-2 antibody but not by control IgG. This Co-IP was specificbecause Bcl-2 co-transfection was necessary. Analysis of other TR3mutants revealed that the C-terminal domain (DC3), but not theN-terminal domain (N168), of TR3/ΔDBD was responsible for binding Bcl-2.The C-terminal fragment DCl (467-536 aa) strongly interacted with Bcl-2,while its deletion from TR3/ΔDBD (TR3/ΔDBD/ΔDC1) largely abolished theinteraction. Furthermore, deletion of a putative amphipathic α-helix(471-488 aa) from TR3/ΔDBD (TR3/ΔDBD/Δ471-488) or mutation of Leu487, acritical amino acid reside for α-helix formation, to Ala(TR3/ΔDBD/L487A) significantly impaired the interaction between TR3/ΔDBDand Bcl-2. Thus, the DC1 region in the TR3 LBD is involved in the Bcl-2interaction. In summary, it was found that the C-terminal domain (DC3),but not the N-terminal domain (N168), of TR3/ΔDBD was responsible forbinding to Bcl-2.

[0131] DC-1 is a 69 residue fragment that corresponds to a portion ofthe ligand-binding domain (LBD) of the nuclear receptor family ofproteins (Mages et al., 1994, Mol Endocrinol 8:1583-1591). LBDs arealpha-helical domains, composed of twelve alpha-helices that arrangethemselves in complex helix bundles (Bourguet et al., 1995, Nature375:377-382). The DC-1 segment of TR3 corresponds to helices 6-9 of LBDs(Mages et al., 1994, supra; Bourguet et al 1995, supra) indicating thatabout ⅔ of DC-1 is alpha helical.

[0132] The 69 amino acid C-terminal fragment (DC1) was able to stronglyinteract with Bcl-2, whereas deletion of DC1 from TR3/(DBD(TR3/(DBD/(DC1) largely abolished its interaction with Bcl-2. However,the interaction of DC1 with Bcl-2 was slightly weaker than that of DC3and Bcl-2. Indeed, the C-terminal portion of DC3 (JK5) also exhibitedslight interaction with Bcl-2.

[0133] To further determine the interaction of DC1 with Bcl-2, a regionof hydrophobic amino acids (HRLGCARGFGDWIDSILA—SEQ ID NO:1) was deletedfrom TR3/ΔDBD, and the resulting mutant (TR3/ΔDBD/Δ471-488) was analyzedfor its interaction with Bcl-2. TR3/ΔDBD/Δ481-488, with eighteen aminoacids from 471 to 488 in the DC1 region deleted from TR3/ΔDBD,TR3/ΔDBD/I483A (with Ile483 replaced with Ala) and TR3/ΔDBD/L487A (withLeu487 replaced with Ala) were analyzed for their interaction with Bcl-2by in vivo Co-IP assay as described above. As compared to TR3/ΔDBD, themutant showed only a very week interaction with Bcl-2. Moreover, asingle hydrophobic amino acid mutation (Ile 483 or Leu 487) largelyabolished the interaction of TR3/ΔDBD and Bcl-2. These data furtherconfirmed the role of DC1 in mediating the interaction between TR3 andBcl-2.

Example 4 Characterization of TR3/Bcl-2 Interaction

[0134] Binding of BH3 domain to Bcl-2 is mediated by a hydrophobic cleftformed by the BH1, BH2, and BH3 region of Bcl-2 (Sattler et al., 1997,Science 275:983-986). To determine whether TR3/ΔDBD bound to the Bcl-2hydrophobic groove, several Bcl-2 mutants with mutations of amino acids,Tyr108, Leu137, or Arg146, critical for the formation of the hydrophobiccleft, were analyzed for their interaction with TR3/ΔDBD. A schematicrepresentation of these mutants is shown in FIG. 3 with the Bcl-2homology (BH) and loop domains and α-helical region indicated. TheseBcl-2 mutants are defective in forming the hydrophobic groove. Mutationsin the Bcl-2 hydrophobic groove abolish Bcl-2 interaction with Bax. TheBcl-2 mutants, Y108KBcl-2, L137ABcl-2 and R146QBcl-2, were analyzed fortheir interaction with GFP-TR3/ΔDBD in 293T cells by Co-IP assay asdescribed in Example 2. Briefly lysates from HEK293T cells transfectedwith GFP-TR3/ΔDBD and the empty vector or the indicated Bcl-2 plasmidwere used. GFP-TR3/ΔDBD (6 μg) was expressed in HEK293T cells with orwithout Bcl-2 (2 μg) in the presence or absence of Bax (2 μg). Lysateswere immunoprecipitated with anti-Bcl-2 antibody. Immunoprecipitates andlysates were examined by Western blotting using the appropriateantibodies. The deletion or point mutations (Y108K, L137A, G145A orR146Q) in Bcl-2 abolished or reduced the interaction with Bax. Incontrast, they were still capable of binding to TR3/ΔDBD. Thus, thehydrophobic cleft of Bcl-2 is not involved in the interaction with TR3,indicating that TR3 interacts with Bcl-2 in a manner that is differentfrom other known Bcl-2-interacting proteins.

[0135] The ability of Bax or Bcl-Gs, a BH3-only Bcl-2-family protein, tocompete with TR3 for binding Bcl-2 was also analyzed byimmunoprecipitation. GFP-DC1 (4 μg) was expressed in HEK293T cells withor without Bcl-2 (2 μg) in the presence or absence of GFP-Bcl-Gs orGFP-Bcl-Gs/L216E (4 μg). Lysates were immunoprecipitated by anti-Bcl-2antibody, followed by Western blotting with anti-GFP or anti-Bcl-2antibodies. The results showed that neither Bcl-Gs nor Bax interferedwith DC1 or TR3/ΔDBD binding to Bcl-2. Rather, these proteinsconsistently enhanced the interaction of Bcl-2 with the TR3 mutants. Incontrast, Bcl-Gs containing a mutation in its BH3 region (Bcl-Gs/L216A),known to prevent binding to Bcl-X_(L)/Bc1-2, did not enhance theinteraction between DC1 and Bcl-2. These results demonstrate that TR3binds Bcl-2 in a manner different from Bcl-Gs and Bax and that theBH3-binding hydrophobic groove in Bcl-2 is not required.

[0136] The above observation that deletion of the BH1, BH2, or BH3domain from Bcl-2 had no effect on its interaction with TR3 suggestedthat the N-terminal portion of the protein was responsible for bindingTR3. Therefore, TR3/ΔDBD was analyzed as to its interaction with afragment of Bcl-2 comprising the first 80 amino acids (Bcl-2/1-80).Co-IP assays demonstrated that Bcl-2/1-80, like the full-length Bcl-2,strongly interacted with TR3/ΔDBD. The Bcl-2/1-80 fragment encompassesthe N-terminal BH4 domain followed by an unstructured loop domain ofapproximately 50 amino acids length. To determine whether the BH4 domainor the loop region was responsible for binding to TR3/ΔDBD, TR3/ΔDBDinteraction with Bcl-2 mutants lacking the BH4 domain (Bcl-2/ΔBH4) orthe loop region (Bcl-2/ΔLoop) was analyzed. Co-IP assays demonstratedthat the Bcl-2/ΔBH4 retained the ability to interact with TR3/ΔDBD,whereas Bcl-2/ΔLoop did not. Thus, the loop region of Bcl-2 is involvedin binding to TR3.

[0137] Deletion of the loop region of Bcl-2 completely blockspaclitaxel-induced apoptosis (Rakesh et al., 1999, Proc Natl Acad Sci96, 3775-3780). To further investigate whether the loop region of Bcl-2is responsible for its interaction with TR3/ΔDBD, TR3/ΔDBD wasco-transfected with the first 80-residue fragment of Bcl-2 including theloop region and BH4 domain. The mutant strongly interacted withTR3/ΔDBD. Deletion of BH4 domain from Bcl-2 did not abolish theinteraction demonstrating that the loop region of Bcl-2 is responsiblefor binding to TR3/ΔDBD.

[0138] To further characterize the interaction between TR3 and Bcl-2, anexperiment was performed to determine whether a BH3-only Bcl-2-familyprotein Bcl-Gs (Guo et al., 2001, J Biol Chem 276:2780-2785) couldaffect the binding of TR3 to Bcl-2. Bcl-2 was co-transfected into 293Tcells with either GFP-DC1 or GFP-Bcl-Gs or a Bcl-Gs mutant and theco-immunoprecipitation (co-IP) was performed as described above. Bcl-Gsis known to bind to the hydrophobic cleft of Bcl-2. Interestingly,incubation of Bcl-Gs did not compete with DC1 for binding Bcl-2.Surprisingly, it enhanced the binding of DC1 to Bcl-2. The enhancingeffect required its binding to Bcl-2 since mutant Bcl-Gs (L216EBcl-Gs)with a mutation in its BH3 domain, which abolishes its ability ofbinding Bcl-2, failed to enhance DC1 binding to Bcl-2. The observationthat Bcl-G enhanced the interaction between DC-1 and Bcl-2 implies thatBcl-G and DC-1 bind different sites on Bcl-2. Similar results wereobtained when BAX was analyzed. Thus, TR3 interacts with Bcl-2 in amanner that is different from that of Bcl-2-family proteins.

[0139] Both LBDs and Bcl-2-family proteins are characterized byplasticity; that is, they undergo conformational reorganization ofhelices upon either binding ligand (LBDs) (Bourguet et al., 1995, supra)or a variety of proteins including Bcl-2-family members (Bcl-2) (Sattleret al., 1997, supra). Bcl-2-family proteins form heterodimers with otherfamily members including Bcl-2 when the alpha-helical BH3 domain of onemember undergoes reorganization to bind the BH3 binding pocket of asecond member (Sattler et al., 1997 supra). Bcl-G very likely interactswith Bcl-2 in this manner to enhance binding of DC-1 . Many proteinsinteract with Bcl-2 possibly through helix-helix interactions. Forexample, the BAG-1 domain, which is a triple helix bundle, binds Bcl-2(Brinknarova et al., 2001, Nat Struct Biol 8:349-352; Reed, 1997, Nature387:773-776). Thus, it is believed that one or more DC-1 helix orhelices interact with Bcl-2 helices to induce a proapoptotic complex.

Example 5 Bcl-2 Mediates TR3 Mitochondrial Localization

[0140] Experiments were performed to determine whether Bcl-2 mediatesTR3 mitochondrial targeting, including confocal microscopy, subcellularfractionation and RNA inhibition.

[0141] A. confocal microscopy—First an experiment was performed todetermine whether the deletion mutant TR3/ΔDBD targets mitochondria viaits interaction with Bcl-2. In confocal microscopy analysis,GFP-TR3/ΔDBD and Bcl-2 were transfected into 293T cells alone ortogether. Cells were then immunostained with anti-Bcl-2 antibodyfollowed by Cy3-conjugated secondary antibody (Sigma) to detect Bcl-2,or with anti-Hsp60 antibody followed by Cy5-conjugated secondaryantibody (Sigma) to detect mitochondria. Bcl-2, TR3/ΔDBD andmitochondria (Hsp60) were visualized using confocal microscopy and thethree images were overlaid. TR3/ΔDBD expressed in 293T cells exhibited adiffuse distribution pattern. However, Bcl-2 colocalized with TR3/ΔDBDwhen they were coexpressed, displaying a distribution pattern overlaidextensively with that of Hsp60. Mutants of TR3 (TR3/ΔDBD/L487A andTR3/ΔDBD/Δ471-488) which did not bind Bcl-2 failed to colocalize withmitochondria.

[0142] B. Subcellular Fractionation—To examine the role of Bcl-2 inmitochondrial targeting of TR3 by an independent method, mitochondriallocalization was also demonstrated by immunoblotting of themitochondria-enriched heavy membrane (HM) fractions. The accumulation ofBcl-2-binding TR3/ΔDBD and non-binding TR3/ΔDBD/Δ471-488 was compared inmitochondria-enriched heavy membrane fractions of HEK293T cellstransfected with or without Bcl-2. The purity of HM preparations wasestablished by assessing the expression of mitochondrial Hsp60, nuclearprotein PARP, and cytosolic/nuclear protein Jun N-terminal kinase (NJK).The heavy membrane fractions were prepared and analyzed for accumulationof TR3/(DBD by Western blotting using anti-GFP antibody. GFP-TR3/ΔDBD orGFP-TR3/ΔDBD/(471-488 (6 mg) and Bcl-2 (2 mg) expression vectors weretransfected into HEK293T cells alone or together. HM fractions wereprepared and analyzed for accumulation of TR3/ΔDBD in mitochondria byimmunoblotting using anti-GFP antibody. The same membrane was alsoblotted with anti-Bcl-2, anti-Hsp60, anti-PARP, or anti-JNK antibody toensure HM purity whole lysate was prepared from cells transfected withTR3/ΔDBD and Bcl-2. TR3/ΔDBD accumulated in the HM fraction (inmitochondria) in the presence of Bcl-2. In contrast, TR3/ΔDBD/Δ471-488was not found in the HM fraction, either in the absence or presence ofBcl-2, a result consistent with the confocal microscopy results above.These results demonstrate that Bcl-2 acts as a receptor for TR3 totarget mitochondria.

[0143] C. Small interfering RNA (siRNA)—To complement these genetransfection experiments, a small interfering (si)RNA approach was usedto determine the effect of suppressing endogenous Bcl-2 expression onTR3 mitochondrial targeting. Briefly, MGC80-3 cells were transfectedwith Bcl-2 siRNA SMARTpool® or control GFP siRNA or left alone. After 48h, lysates were prepared and assayed by immunoblotting using anti-Bcl-2and anti-β-actin antibodies. In MGC80-3 gastric cancer cells, a tumorcell line in which TR3 was reported to target mitochondria in responseto specific apoptotic stimuli, Bcl-2 expression was almost completelyinhibited by Bcl-2-specific siRNA, but not by GFP siRNA control. Bothconfocal microscopy and immunoblotting of HM fractions revealed thatendogenous TR3 targeted mitochondria in MGC80-3 cells treated with3-Cl-AHPC. However, transfection of Bcl-2 siRNA, but not control GFPsiRNA, largely abolished mitochondrial targeting of TR3. 3-Cl-AHPC stillinduced expression of TR3 and translocation of TR3 from the nucleus tocytosol, but TR3 failed to target mitochondria in the absence of Bcl-2.Similarly, inhibition of endogenous Bcl-2 expression by Bcl-2 antisenseoligonucleotides impaired TR3 mitochondrial targeting in H460 lungcancer cells.

[0144] The next approach involved using a fragment of Bcl-2 consistingof the TR3 interaction domain (loop region) to identify whether it couldact in a dominant-negative fashion to inhibit TR3 mitochondrialtargeting in LNCaP cells. Cells transiently transfected with theGFP-Bcl-2/1-90 mutant comprising of the first 90 N-terminal amino acidswere treated with TPA to induce expression of TR3 and mitochondrialtargeting of the endogenous TR3. In cells transfected withGFP-Bcl-2/1-90, TR3 failed to target mitochondria, displaying a diffusecytosolic distribution pattern in contrast to non-transfected cells inthe same culture dish. Thus, Bcl-2/1-90 inhibits TR3 mitochondrialtargeting, probably by competing with endogenous Bcl-2 for binding toTR3. Together, these results demonstrate that Bcl-2 acts as a receptorfor TR3, and is responsible for its mitochondrial targeting.

Example 6 Bcl-2 Mediates TR3-induced Cytochrome c Release

[0145] The involvement of TR3/Bcl-2 interaction in TR3-inducedcytochrome c release was also studied. GFP-TR3/(DBD (4 μg) and Bcl-2 (2μg) were transfected into 293T cells alone or together. GFP-TR3/ΔDBDwere also co-expressed with Bcl-2/ΔTM or Bcl-2/Y108K (2 μg). Cells wereimmunostained with anti-cytochrome c (cyt c) or anti-Bcl-2 antibodyfollowed by Cy5 conjugated secondary antibody (Sigma) to detectcytochrome c, or with anti-Hsp60 followed by Cy3-conjugated secondaryantibody (Sigma) to detect mitochondria or anti-Bcl-2 antibody followedby Cy5-conjugated secondary antibody to detect Bcl-2. Cytochrome c,TR3/(DBD, and mitochondria (Hsp60) were visualized using confocalmicroscopy, and images for TR3/(DBD and Hsp60 were overlaid.Approximately 75% of the TR3/ΔDBD and Bcl-2 colocalized cells displayedvarious levels of diffuise cyt c staining. In the absence of Bcl-2cotransfection, TR3/ΔDBD expression did not cause any release ofcytochrome c from mitochondria as determined by confocal microscopyanalysis, which showed punctate cytochrome c staining. Nor did transientexpression of Bcl-2 alone. However, cotransfection of Bcl-2 andTR3/ΔDBD, resulted in colocalization of TR3/ΔDBD and Bcl-2 andsignificant release of cytochrome c from mitochondria. Cyt c releaseinvolved mitochondrial localization of TR3/ΔDBD and Bcl-2, because itdid not occur upon co-transfection of TR3/ΔDBD with Bcl-2/ΔTM, a Bcl-2mutant lacking the ability to target mitochondria. Interestingly,co-expression of TR3/ΔDBD and Bcl-2/Y108K did not induce cyt c release,although they colocalized. This result suggests that the interactionbetween TR3/ΔDBD and Bcl-2 is insufficient for inducing cyt c release.

[0146] TR3/ΔDBD targets mitochondria in NCI-H460 cells, which expresshigh levels of Bcl-2 (Lu et al., 2001, Cancer Chemother Pharmacol 47:170-178). To determine whether Bcl-2 is involved in mitochondrialtargeting of TR3, GFP-TR3/ΔDBD was transfected into Calu-6 lung cancercells, which express low levels of Bcl-2. GFP-TR 3/ΔDBD and Bcl-2 weretransfected into Calu-6 lung cancer cells alone or together. Cells werethen immunostained with anti-Bcl-2 antibody followed by Cy3-conjugatedsecondary antibody to detect Bcl-2, or with anti-Hsp60 antibody followedby Cy5-conjugated secondary antibody to detect mitochondria. Bcl-2,TR3/(DBD and mitochondria (Hsp60) were visualized using confocalmicroscopy. Unlike its exclusive localization in mitochondria inNCI-H460 cells, TR3/ΔDBD was diffusely distributed in Calu-6 cells, buttargeted mitochondria when Bcl-2 was co-transfected. Moreover,cytochrome c was released from mitochondria of TR3/ΔDBD-Bcl-2cotransfected Calu-6 cells. Thus, Bcl-2 is involved in TR3/ΔDBD totargeting of mitochondria and the subsequent induction of cytochrome crelease.

Example 7 Effect of Various TR3 Mutants on Apoptotic Potential of Bcl-2

[0147] DAPI staining was used to study the apoptotic effect of TR3/Bcl-2interaction. 293T cells were transfected with GFP-TR3/ΔDBD alone ortogether with Bcl-2. After 36 h, nuclei were stained by DAPI and nuclearfragmentation and chromatin condensation were identified. Cells werealso stained with anti-Bcl-2 antibody, followed by TRITC-conjugatedsecondary antibody (Sigma). Bcl-2 and GFP-ΔDBD expression and nuclearmorphology were visualized by fluorescence microscopy, and the twoimages were overlaid. Transfected TR3/ΔDBD did not cause any nuclearfragmentation or condensation in 293T cells. But, when Bcl-2 wascotransfected, a significant amount of transfected cells underwentapoptosis. For example, approximately 4% of cells transfected witheither TR3/ΔDBD or Bcl-2 were apoptotic, compared to 35% of celltransfected with both. The pro-apoptotic effect of Bcl-2 seen uponco-expression, was specific to TR3, because Bax-induced apoptosis waseffectively prevented by Bcl-2 co-expression. Cotransfection of Bcl-2,however, prevented BAX-induced apoptosis in these cells (see FIG. 4).The effect of Bcl-2 on apoptotic potential of TR3/ΔDBD in lung cancerand breast cancer cells was also investigated. In these experiments,Bcl-2 was cotransfected into the 293T, MCF-7, or Calu-6 cell lines withor without TR3(DBD or BAX. After 36 h, nuclei were stained by DAPI.Cells displaying nuclear condensation or fragmentation were scored. Inthe absence of Bcl-2, expression of TR3/ΔDBD did not show any apoptoticeffect in MCF-7 breast cancer and Calu-6 lung cancer cells. However,co-expression of Bcl-2 resulted in strong induction of apoptosis in bothcell lines. Thus, the TR3-Bcl-2 interaction is involved in the inductionof cytochrome c release and apoptosis.

[0148] The effect of inhibiting endogenous Bcl-2 expression on theTR3-dependent apoptosis in MGC80-3 cells was next analyzed. Treatment ofcontrol GFP-siRNA-transfected cells with 3-Cl-AHPC resulted inapoptosis, consistent with prior studies showing that this agent inducesTR3 expression and mitochondrial targeting. However, 3-Cl-AHPC-inducedapoptosis was suppressed by more than half (about 60%) in Bcl-2siRNA-transfected cells. Similar results were obtained in H460 lungcancer cells. Moreover, expression of the Bcl-2/1-90 protein, whichinhibited TR3 mitochondrial targeting, also suppressed TR3-dependentapoptosis induced by apoptotic stimuli, such as TPA and 3-Cl-AHPC, inLNCaP cells. Thus, Bcl-2 can manifest a pro-apoptotic phenotype insettings where TR3 is expressed and targets to mitochondria.

[0149] To extend some of these studies using established tumor celllines to normal cells, experiments were performed using primary culturesof peripheral blood lymphocytes (PBLs). TR3 subcellular localization wasstudied by both confocal microscopy and subcellular fractionationapproaches. For microscopy experiments, freshly isolated PBLs weretransfected with GFP-TR3, then treated with phorbol ester TPA and thecalcium ionophore ionomycin, which induce TR3-dependent apoptosis ofT-lymphocytes. Without treatment, GFP-TR3 mainly resided in the nucleus.After treatment, GFP-TR3 was found in the cytoplasm, colocalizing withco-transfected DsRed2-Mito, a red fluorescent protein (RFP) fuised witha mitochondria-targeting sequence. Subcellular fractionation experimentsrevealed that TPA/ionomycin treatment induced accumulation of endogenousTR3 in HM fractions. Interestingly, this treatment also altered themigration of TR3 protein, suggesting a possible post-translationalmodification. Thus, both transfected and endogenous TR3 also targets tomitochondria in primary lymphocytes.

[0150] The role of Bcl-2 in TR3-dependent apoptosis in PBLs was alsostudied. Treatment with TPA/ionomycin induced extensive apoptosis ofPBLs, which was partially inhibited by transfecting Bcl-2 antisenseoligonucleotides or TR3 siRNA. The role of Bcl-2 in TR3/ΔDBD targetingto mitochondria and in TR3/ΔDBD-induced apoptosis in PBLs was studied intransfection experiments. In these experiments, GFP-TR 3/ΔDBDcolocalized extensively with DsRed2-Mito and potently induced PBLapoptosis, as determined by annexin V-staining. In contrast,TR3/ΔDBD-induced apoptosis was considerably suppressed by Bcl-2antisense oligonucleotides in PBLs. Thus, endogenous Bcl-2 contributesto TR3-dependent apoptosis in primary lymphocytes.

Example 8 Domains in TR3 and Bcl-2 Involved in their Apoptotic Activity

[0151] To characterize the pro-apoptotic mechanism of Bcl-2 in thesetting of TR3-induced apoptosis, various TR3 mutants and Bcl-2 mutantswere co-expressed in HEK293T cells. TR3 mutants were transfected into293T cells together with or without Bcl-2 expression vector or withBcl-2 mutants. After 36 h, nuclei were stained by DAPI. Cells displayingnuclear condensation or fragmentation were scored. Expression of DC3 andDC1 that showed strong interaction with Bcl-2 potently induced apoptosisof 293T cells, when Bcl-2 was co-expressed (see FIG. 18). In contrast,coexpression of TR3/ΔDBD/ΔDC1 that failed to interact with Bcl-2 did notresult in apoptosis of 293T cells, indicating that the DC1 region iscapable of inducing apoptosis. DC3 also induced apoptosis, consistentwith the ability of these TR3 fragments to bind Bcl-2. The role of theDC1 in inducing the apoptotic potential of Bcl-2 was also demonstratedby the observation that coexpression of either TR3/ΔDBD/ΔDC1,TR3/ΔDBD/Δ471-488, TR3/ΔDBD/L487A or TR3/ΔDBD/I483A and Bcl-2 failed toconfer the apoptotic potential of Bcl-2.

[0152] The role of various Bcl-2 mutants in mediating apoptosisinduction by TR3 was next studied. Bcl-2 effectively suppressedapoptosis induced by Bax expression in HEK293T cells, indicating thatBcl-2 is a potent anti-apoptotic molecule with respect to Bax-inducedapoptosis. The hydrophobic cleft of Bcl-2 is involved in itsanti-apoptotic effect through its interaction with BH3 domain ofpro-apoptotic molecules (Reed 1997, Semin Hematol 34:9-19; Reed 1997,Nature 387:773-776). To investigate whether the BH3 domain-binding motifof Bcl-2 is also involved in its apoptotic effect, various Bcl-2 mutantsincluding Y108K Bcl-2, with a mutation in the BH3 domain-binding cleft,L137A, and G145A (all with impaired interaction with bax), werecotransfected together with TR3/ΔDBD. More specifically, expressionvectors for Bcl-2 or BH3 domain point mutant of Bcl-2 or other mutantswere co-transfected together with or without TR3/ΔDBD or BAX or GFP into293T cells, and after 36 hours, nuclei were stained by DAPI and thepercentage of GFP-positive cells with nuclear condensation orfragmentation was scored (FIG. 6). Y108K Bcl-2 could not bind to BAX andfailed to prevent BAX-induced apoptosis in 293T cells (FIG. 6). Bcl-2mutations (Y108K, L137A, G145A) that impaired its interaction with Bax,abolished the Bcl-2 inhibitory effect on Bax-induced apoptosis,consistent with previous observations that the hydrophobic cleft ofBcl-2 is essential for its anti-apoptotic effect. Interestingly, Y108Kwas unable to induce apoptosis when it was cotransfected with TR3/ΔDBD,although it is capable of binding to TR3/ΔDBD. Thus, an intact BH3domain-binding motif in Bcl-2 is involved in its pro-apoptotic effect.In contrast to the inhibitory effect of Bcl-2 on Bax-induced apoptosisTR3/ΔDBD-induced apoptosis was augmented by co-expression of Bcl-2 inHEK293T cells. As expected, co-expression of TR3/ΔDBD with Bcl-2/ΔLoopdid not induce cell death, consistent with the inability of this Bcl-2mutant to bind TR3.

[0153] Mitochondrial targeting of TR3 has been previously identified asbeing involved in its apoptotic effect. To study whether mitochondriallocalization of the TR3/ΔDBD/Bcl-2 complex was involved in theirapoptotic effect, TR3/ΔDBD was cotransfected with Bcl-2/ΔTM, a Bcl-2lacking its transmembrane domain. Coexpression of TR3/ΔDBD and Bcl-2/ΔTMdid not result in apoptosis of 293T cells. Thus, mitochondriallocalization of TR3/ΔDBD/Bcl-2 complex is involved in this apoptoticeffect.

[0154] Experiments to delineate the structure-function relationsinvolved in the pro-apoptotic effect of Bcl-2 in the context ofTR3-induced apoptosis showed the following: Though capable of bindingTR3/ΔDBD, mutants of Bcl-2 lacking the membrane-anchoring TM domain, theBH1 domain, BH2 domain, or BH3 domain were incapable of inducingapoptosis when co-expressed with TR3/ΔDBD. Similarly, a BH3 mutant ofBcl-2 (Y108K) also failed to induce apoptosis in collaboration with TR3,though it retained the ability to bind TR3. This observation isconsistent with the data above, showing that Bcl-2/Y108K failed toinduce cyt c release when co-expressed with TR3/ΔDBD. Moreover,mutations of the BH3-binding pocket of Bcl-2, L137A and G145A, whichabrogated the ability of Bcl-2 to suppress apoptosis induced by Bax,retained the ability to promote apoptosis when co-expressed withTR3/ΔDBD. Thus, an intact hydrophobic groove in Bcl-2 is involved in itsanti-apoptotic activity but not its pro-apoptotic activity,demonstrating a structural distinction between these two opposingphenotypes of Bcl-2.

Example 9 Mitochondrial Targeting of TR3 in Neurons

[0155] To further investigate TR3 targeting of mitochondria, rat primaryneurons were treated with the dopaminergic neurotoxin1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). MPTP inducesParkinson's disease in animals. Rat primary dopaminergic neurons weretreated with or without 100 μM MPTP for one (1) hour, then immunostainedwith anti-TR3 antibody followed by Cy3-conjugated secondary antibody(Geneka) to detect endogenous TR3, or with anti-Hsp60 antibody followedby Cy5-conjugated secondary antibody (Sigma) to detect mitochondria.Neurons were identified by staining with neuron marker. TR3 andmitochondria (Hsp60) were visualized using confocal microscopy.Endogenous TR3 was found exclusively in the cytoplasm and colocalizedwith mitochondria in MPTP-treated neurons. TR3 was found only in thenucleus of non-treated neurons. Thus, TR3 mitochondrial localizationplays a role in mediating the effect of dopaminergic neurotoxin inneurons.

[0156] The effect of phorbol ester (TPA) and calcium ionophore (A23187)on TR3 subcellular localization in primary hippocampal neurons was alsoexamined. Rat primary hippocampal neurons were treated with or without100 ng/ml TPA and 10 μM A23187 for 1 hour. The neurons were thenimmunostained with anti-TR3 antibody (Geneka) followed by Cy3-conjugatedsecondary antibody (Geneka) to detect endogenous TR3, or with anti-Hsp60antibody followed by Cy5-conjugated secondary antibody (Sigma) to detectmitochondria. TR3 and mitochondria (Hsp60) were visualized usingconfocal microscopy. Treatment with TPA and A23187 caused TR3relocalization from the nucleus to the mitochondria. Thus, TR3mitochondrial localization is associated with neuronal death.

[0157] To determine whether additional molecules could induce apoptosisin a manner similar to that resulting from the interaction between TR3and Bcl-2, another member of the Bcl-2-family, the apoptosis antagonistBcl-X_(L), was studied, as described below.

Example 10 TCTP Binds Bcl-X_(L) and Induces Apoptosis

[0158] Bcl-X_(L) cDNA was constructed into the pGilda yeast two-hybridvector. Using pGilda-Bcl-X_(L) as bait, a human testis cDNA library wasused in yeast two-hybrid screening to screen for potential Bcl-X_(L)interacting proteins. pGilda-Bcl-X_(L) and a reporter plasmid pSH18-34were co-transfected into EGY48 yeast cells. The tranfectant yeast cellswere then transfected in large scale with yeast two-hybrid library cDNAplasmids. Positive clones were identified through selection inLeucine-deficient media. A total of 95 positive colonies wereidentified. These positive clones were further examined for a secondreporter expression by β-gal filter assays. Among these clones, 29 ofthem tested positive. These clones were further examined by matingassays to confirm the interaction with Bcl-X_(L).Ten positive cloneswere isolated and sequenced to reveal their identity. Two of them encodecDNAs identical to human Translationally-Controlled Tumor Protein(TCTP).

[0159] TCTP is a calcium-binding protein with no previous identifiedrole in apoptosis regulation. It has been discovered that TCTP interactswith Bcl-X_(L). HEK293T cells were transfected with pcDNA3-HA vectorcontaining Bcl-X_(L) together with GFP or GFP-TCTP. Cell lysates wereimmunoprecipitated with polyclonal anti-GFP antibody. Theimmunoprecipitates or the lysates were blotted with anti-HA or anti-GFPantibodies, respectively.

[0160] Fluorescence microscopy demonstrated the co-localization of theseproteins. GFP, RFP, GFP-TCTP and RFP-Bcl-X_(L) plasmids were transfectedinto cos-7 cells. Images were photographed with a confocal microscope.Furthermore, Bcl-X_(L) recruits TCTP from cytosolic location to themitochondria.

[0161] An interesting and unique functional property of TCTP, however,is that it not only induces apoptosis when over-expressed in cells,similar to many pro-apoptotic proteins, it also kills cells better whenBcl-X_(L) is co-expressed with it. GFP or GFP-TCTP plasmids wereco-transfected with RFP-Bcl-X_(L) or RFP-Bcl-X_(L) (ΔTM) plasmids intocos-7 cells. Cells were fixed and stained with DAPI 48 hours aftertransfection (n≧200 total cells evaluated) and the percentage ofapoptotic cells was calculated (mean±SD, n=3) (FIG. 7). More apoptosisof MCF7 cells was observed when TCTP was expressed together withBcl-X_(L) than without Bcl-X_(L). This indicates that TCTP's interactionwith Bcl-X_(L) converts Bcl-X_(L) from an anti-apoptotic to apro-apoptotic protein.

[0162] Additionally, it was shown that the BH3 domain of Bcl-X_(L) wasinvolved in the TCTP induced apoptosis. GFP-TCTP plasmids wereco-transfected with RFP-Bcl-X_(L), RFP-Bcl-X_(L) (F97A/E98A) orRFP-Bcl-X_(L) (G94A) plasmids into cos-7 cells. Cells were fixed andstained with DAPI 48 hours after transfection (n≧200 total cellsevaluated) and the percentage of apoptotic cells was calculated(mean±SD, n=3). Results are shown in FIG. 8.

Example 11 Bcl-2 Undergoes a Conformational Change Upon TR3 Binding

[0163] Pro-apoptotic Bcl-2-family proteins Bax and Bak have been shownto undergo conformational changes in association with their conversionfrom latent to active killer proteins. Therefore the possibility that aconformational change might be involved in the conversion of Bcl-2 fromanti-apoptotic to pro-apoptotic function was explored. To this end, theeffects of TR3 on binding of Bcl-2 to various anti-Bcl-2 antibodiesrecognizing different epitopes Were compared. Antibody binding to Bcl-2was measured by imnmunofluorescence using flow cytometry (See FIGS.9a-f) or by immunoprecipitation.

[0164] First, HEK293T cells were transfected with Bcl-2 (5 μg), togetherwith GFP-TR3/ΔDBD or the control GFP vector (5 μg) for 14 h., andimmunostaining was performed on fixed and permeabilized cells usingrabbit polyclonal antibody raised against the whole Bcl-2 protein, mousemonoclonal antibody directed against the Bcl-2 BH3-binding pocket, orpolyclonal antibody raised against the Bcl-2 BH3 domain followed bySRPD-conjugated secondary antibody (Southern Biotech). The transfectedcells were identified by their green fluorescence by flow cytometricanalysis, and the intensity of Bcl-2 immunofluorescence was comparedbetween the GFP-positive and GFP-negative cells. Bcl-2immunofluorescence was undetectable in control GFP-expressing cells bystaining with the antibody directed against the Bcl-2 BH3 Domain butdramatically increased in GFP-TR3/ΔDBD -expressing cells (FIG. 9a and 9d), suggesting increased availability of this epitope in Bcl-2 uponTR3/ΔDBD co-expression. In contrast, the immunofluorescence obtained bystaining with the antibody directed against the BH3-binding pocket wasreduced by co-expression of GFP-TR3/ΔDBD, suggesting decreasedavailability of this epitope (FIG. 9b and 9 e). These alterations inbinding of epitope-specific antibodies to Bcl-2 in response toGFP-TR3/ΔDBD co-expression were not due to changes in Bcl-2 levels,because GFP-TR3/ΔDBD co-expression did not result in altered Bcl-2immunofluorescence when the Bcl-2 antibody raised against the wholeprotein was used. In addition, immunoblotting analysis and BD cytometricbead assays revealed equivalent levels of Bcl-2 protein in the presenceof GFP or GFP-TR3/ΔDBD. Also, TR3/ΔDBD co-expression did not modifybinding of these epitope-specific antibodies to Bcl-2/ΔLoop, asdetermined by immunofluorescence measurements using flow cytometr. ThisTR3/ΔDBD-induced change in Bcl-2 conformation was also observed in PBLsin addition to HEK293T cells (See FIGS. 9a-f).

[0165] Second, the effects of TR3/ΔDBD on Bcl-2 conformation werestudied by immunoprecipitation assays. These experiments showed thatco-expression of TR3/ΔDBD reduced binding of Bcl-2 by the antibodydirected against the BH3-binding pocket, while enhancing binding ofBcl-2 by the antibody directed against the BH3 domain. In contrast,TR3/ΔDBD did not affect the immunoprecipitation efficiency of the Bcl-2antibody raised against the whole protein. Together, these resultsdemonstrated that TR3 binding induced a conformational change, resultingin exposure of its BH3 domain.

[0166] Pro-apoptotic BH3-only members of the Bcl-2 family induceapoptosis by binding to other Bcl-2 family members through their BH3domains. Therefore, it was of interest to determine whether TR3 bindingalters the ability of Bcl-2 to bind Bcl-X_(L) or Bak, as measured byCo-IP assays. At least when assessed in detergent-containing lysates ofcells by Co-IP, Bcl-2 bound Bcl-X_(L) and Bak independently of TR3. Toaddress the possibility that Bcl-2 bound differently to Bcl-X_(L) andBak in the presence of TR3/ΔDBD, two Bcl-2 mutants were analyzed.Bcl-2/L137A, a BH3-binding pocket mutant that retained killing activityin the presence of TR3/ΔDBD, interacted with Bcl-X_(L) and Bak only whenTR3/ΔDBD was co-expressed. In contrast, binding to Bcl-X_(L) and Bak ofthe Bcl-2/Y108K BH3 domain mutant was not modulated by co-expression ofTR3. Thus, TR3 binding may result in altered association of Bcl-2 withother members of the Bcl-2 family. Moreover, the observation thatBcl-2/L137A, but not Bcl-2/Y108K, was capable of killing cells incollaboration with TR3/ΔDBD suggested that exposure of the BH3 domain ofBcl-2 may be responsible for the conversion of Bcl-2 to a pro-apoptoticmolecule.

[0167] The above data suggested that Bcl-2, upon TR3 binding, inducesapoptosis through its BH3 domain. BH3-only proteins exert theirapoptotic effects through either Bax or Bak. The involvement of Bax andBak in Bcl-2-dependent apoptosis induced by TR3 was therefore examined.Co-transfection of TR3/ΔDBD and Bcl-2 resulted in a similar degree ofapoptosis in HCT116 cells and HCT116 cells lacking Bax (HCT 116Bax-^(−/−)), suggesting that expression of Bax was not crucial. This wasalso supported by the observation that H460 cells, which underwentextensive apoptosis in response to 3-Cl-AHPC, expressed only tracelevels of Bax.

[0168] To determine whether Bak, which was highly expressed in H460cells, played a role in Bcl-2-dependent apoptosis induced by TR3, theeffects of Bak siRNA transfection was examined. Significant reductionsof Bak protein were observed when H460 cells were transfected with BaksiRNA but not control siRNA, correlating with significant repression ofTR3-dependent 3-Cl-AHPC-induced apoptosis. Thus, Bcl-2-dependentapoptosis induced by TR3 depends on multidomain pro-apoptoticBcl-2-family proteins such as Bak.

VI. Methods for Screening Peptides, Analogs, and Small Molecules thatModulate Bcl-2-Family Member Protein Activity

[0169] The assays described above are designed to identify compoundsthat interact with (e.g., bind to) Bcl-2, Bcl-X_(L), and other membersof the Bcl-2-family of proteins, and modify their ability to regulateapoptosis. This regulation may be by mimicking TR3, by inducing anequivalent conformation change, by enhancing the TR3 effect or byinhibiting the TR3 effect.

[0170] The compounds which may be screened include, but are not limitedto peptides, fragments thereof, and other organic compounds (e.g.,peptidomimetics) that bind to the Bcl-2-family member and either mimicthe activity triggered by the natural regulatory ligand (e.g., TR3 andTCTP), enhance the activity triggered by the natural regulatory ligandor inhibit the activity triggered by the natural ligand; as well aspeptides, antibodies or fragments thereof, and other organic compoundsthat mimic the binding domain of the Bcl-2-family member and bind to and“neutralize” natural ligand.

[0171] Such compounds may include, but are not limited to, peptides suchas, for example, soluble peptides, including but not limited to membersof random peptide libraries; (see, e.g., Lam et al., 1991, Nature354:82-84; Houghten et al., 1991, Nature 354:84-86), and combinatorialchemistry-derived molecular libraries made of D- and/or L-configurationamino acids, phosphopeptides (including, but not limited to, members ofrandom or partially degenerate, directed phosphopeptide libraries; see,e.g., Songyang et al., 1993, Cell 72:767-778), antibodies including, butnot limited to, polyclonal, monoclonal, humanized, anti-idiotypic,chimeric or single chain antibodies, and FAb, F(ab′)₂ and FAb expressionlibrary fragments, and epitope-binding fragments thereof, and smallorganic or inorganic molecules.

[0172] Computer modeling and searching technologies permitidentification of compounds, or the improvement of already identifiedcompounds, that can modulate Bcl-2-family member activity. Havingidentified such a compound or composition, the active sites or regionsare identified. The active site can be identified using methods known inthe art including, for example, from the amino acid sequences ofpeptides, from the nucleotide sequences of nucleic acids, or from studyof complexes of the relevant compound or composition with its naturalligand. In the latter case, chemical or X-ray crystallographic methodscan be used to find the active site by finding where on the factor thecomplexed ligand is found. Next, the three dimensional geometricstructure of the active site is determined. This can be done by knownmethods, including X-ray crystallography, which can determine a completemolecular structure. On the other hand, solid or liquid phase NMR can beused to determine certain intra-molecular distances. Any otherexperimental method of structure determination can be used to obtainpartial or complete geometric structures. The geometric structures maybe measured with a complexed ligand, natural or artificial, which mayincrease the accuracy of the active site structure determined.

[0173] If an incomplete or insufficiently accurate structure isdetermined, the methods of computer based numerical modeling can be usedto complete the structure or improve its accuracy. Any recognizedmodeling method may be used, including parameterized models specific toparticular biopolymers such as proteins or nucleic acids, moleculardynamics models based on computing molecular motions, statisticalmechanics models based on thermal ensembles, or combined models. Formost types of models, standard molecular force fields, representing theforces between constituent atoms and groups, are necessary, and can beselected from force fields known in physical chemistry. The incompleteor less accurate experimental structures can serve as constraints on thecomplete and more accurate structures computed by these modelingmethods.

[0174] Finally, having determined the structure of the active site,either experimentally, by modeling, or by a combination, candidatemodulating compounds can be identified by searching databases containingcompounds along with information on their molecular structure. Such asearch seeks compounds having structures that match the determinedactive site structure and that interact with the groups defining theactive site. Such a search can be manual, but is preferably computerassisted. These compounds found from this search are potentialBcl-2-family member modulating compounds.

[0175] Alternatively, these methods can be used to identify improvedmodulating compounds from an already known modulating compound orligand. The composition of the known compound can be modified and thestructural effects of modification can be determined using theexperimental and computer modeling methods described above applied tothe new composition. The altered structure is then compared to theactive site structure of the compound to determine if an improved fit orinteraction results. In this manner systematic variations incomposition, such as by varying side groups, can be quickly evaluated toobtain modified modulating compounds or ligands of improved specificityor activity.

[0176] Further experimental and computer modeling methods useful toidentify modulating compounds based upon identification of the activesites of Bcl-2, Bcl-X_(L), and related proteins will be apparent tothose of skill in the art.

[0177] Examples of molecular modeling systems are the CHARMM and QUANTAprograms (Polygen Corporation, Waltham, Mass.). CHARMM performs theenergy minimization and molecular dynamics functions. QUANTA performsthe construction, graphic modeling and analysis of molecular structure.QUANTA allows interactive construction, modification, visualization, andanalysis of the behavior of molecules with each other.

[0178] A number of articles review computer modeling of drugsinteractive with specific-proteins, such as Rotivinen et al., 1988, ActaPharm Fennica 97:159-166; McKinaly and Rossmann, 1989, Annu RevPharmacol Toxiciol 29:111-122; Perry and Davies, OSAR: QuantitativeStructure-Activity Relationships in Drug Design, pp. 189-193, Alan R.Liss, Inc. (1989); Lewis and Dean, 1989, Proc R Soc Lond 236:125-140 and141-162; and, with respect to a model receptor for nucleic acidcomponents, Askew et al., 1989, J Am Chem Soc 111:1082-1090. Othercomputer programs that screen and graphically depict chemicals areavailable from companies such as BioDesign, Inc. (Pasadena, Calif),Allelix, Inc. (Mississauga, Ontario, Canada), and Hypercube, Inc.(Cambridge, Ontario).

[0179] One could also screen libraries of known compounds, includingnatural products or synthetic chemicals, and biologically activematerials, including proteins, for compounds which exhibit bindingproperties similar to those of TR3, such as lack of competition withBcl-G, or enhanced binding in the presence of Bcl-G.

[0180] One could also screen libraries of known compounds, includingnatural products or synthetic chemicals, and biologically activematerials, including proteins, for compounds which enhance the activityof or binding of TR3 to Bcl-2.

[0181] Once identified, these compounds can be subjected to assays suchas those described in the examples to identify whether the compoundsincrease apoptosis or decrease apoptosis in cells.

[0182] Compounds identified via assays such as those described hereinmay be useful, for example, in inducing or inhibiting apoptosis.

VII. In Vitro Screening Assays for Compounds that Bind to Bcl-2-FamilyMember Proteins

[0183] In vitro systems may be designed to identify compounds capable ofinteracting with (e.g., binding to) Bcl-2-family members. Compoundsidentified may be useful, for example, in modulating the activity ofwild type and/or mutant Bcl related proteins; may be useful inelaborating the biological fumction of the Bcl related proteins; may beutilized in screens for identifying compounds that disrupt normalBcl-2-family member interactions; or may in themselves disrupt suchinteractions.

[0184] The principle of the assays used to identify compounds that bindto the Bcl-2-family member involves preparing a reaction mixture of theprotein and the test compound under conditions and for a time sufficientto allow the two components to interact and bind, thus forming a complexwhich can be removed and/or detected in the reaction mixture.

[0185] The screening assays can be conducted in a variety of ways. Forexample, one approach would involve anchoring the Bcl-2 related protein,polypeptide, peptide or fusion protein or the test substance to a solidphase, and detecting complexes of Bcl-2 related protein bound to testcompounds anchored on the solid phase. In one embodiment, the Bcl-2related reactant may be anchored to a solid surface and the testcompound, which is not anchored, may be labeled, either directly orindirectly. Bound compound(s) could then be detected by various methodssuch as mass spectrometry after elution from the bound protein. Inanother embodiment, the binding specificity of test compounds can betested using a competition assay as follows: a) Bcl-2 or Bcl-X_(L) isanchored to a solid phase; b) immobilized Bcl-2 or Bcl-X_(L) isincubated with TCTP or TR3 labeled with a fluorescent tag or otherreporter molecule, in the presence or absence of compounds being tested;c) after incubation under suitable conditions, the solid phase is washedto remove unbound reactants; d) the amount of labeled TCTP or TR3 boundto the solid phase is measured for each reaction; and e) the amount oflabeled TCTP or TR3 bound in the presence of various test compounds iscompared with the amount of labeled TCTP or NR3 bound in the absence oftest compounds, and the ability of each test compound to compete forBcl-2 or Bcl-X_(L) binding sites is determined.

[0186] In practice, microtiter plates may conveniently be utilized asthe solid phase. The anchored component may be immobilized bynon-covalent or covalent attachments. Non-covalent attachment may beaccomplished by simply coating the solid surface with a solution of theprotein and drying. Alternatively, an immobilized antibody, preferably amonoclonal antibody, specific for the protein to be immobilized may beused to anchor the protein to the solid surface. The surfaces may beprepared in advance and stored.

[0187] In order to conduct the assay, the nonimmobilized component isadded to the coated surface containing the anchored component. After thereaction is complete, unreacted components are removed (e.g., bywashing) under conditions such that any complexes formed will remainimmobilized on the solid surface. The detection of complexes anchored onthe solid surface can be accomplished in a number of ways. Where thepreviously nonimmobilized component is pre-labeled, the detection oflabel immobilized on the surface indicates that complexes were formed.Where the previously nonimmobilized component is not pre-labeled, anindirect label can be used to detect complexes anchored on the surface;e.g., using a labeled antibody specific for the previouslynonimmobilized component (the antibody, in turn, may be directly labeledor indirectly labeled with a labeled anti-Ig antibody).

[0188] Alternatively, a reaction can be conducted in a liquid phase, thereaction products separated from unreacted components, and complexesdetected, e.g., using an immobilized antibody specific for the Bclrelated protein, polypeptide, peptide or fusion protein or the testcompound to anchor any complexes formed in solution, and a labeledantibody specific for the other component of the possible complex todetect anchored complexes.

[0189] Alternatively, cell-based assays can be used to identifycompounds that interact with Bcl-2-family members. To this end, celllines that express Bcl related proteins, or cell lines (e.g., COS cells,CHO cells, fibroblasts, etc.) that have been genetically engineered toexpress Bcl-2 related proteins (e.g., by transfection or transduction ofDNA) can be used.

VIII. Assays for Intracellular Proteins that Interact with theBcl-2-Family Members, TR3, and TCTP

[0190] Any method suitable for detecting protein-protein interactionsmay be employed for identifying transmembrane proteins or intracellularproteins that interact with Bcl-2 or related proteins. Among thetraditional methods which may be employed, are co-immunoprecipitation,crosslinking and co-purification through gradients or chromatographiccolumns of cell lysates or proteins obtained from cell lysates orrecombinantly produced to identify proteins in the lysate that interactwith the Bcl related protein. For these assays, the Bcl relatedcomponent used can be a full-length, a soluble derivative lacking themembrane-anchoring region, a peptide corresponding to a binding domainor a fusion protein containing the binding domain. Once isolated, suchan intracellular protein can be identified and can, in turn, be used, inconjunction with standard techniques, to identify proteins with which itinteracts. For example, at least a portion of the amino acid sequence ofan intracellular protein which interacts with the Bcl related proteincan be ascertained using techniques well known to those of skill in theart, such as via the Edman degradation technique. (See, e.g., Creighton,Proteins: Structures and Molecular Principles, W.H. Freeman & Co., N.Y.(1983) pp.34-49). The amino acid sequence obtained may be used as aguide for the generation of oligonucleotide mixtures that can be used toscreen for gene sequences encoding such intracellular proteins.Screening may be accomplished, for example, by standard hybridization orPCR techniques. Techniques for the generation of oligonucleotidemixtures and the screening are well-known. (See, e.g., Ausubel supra,and PCR Protocols: A Guide to Methods and Applications, Innis, M. etal., eds. Academic Press, Inc., New York (1990)).

[0191] Additionally, methods may be employed which result in thesimultaneous identification of genes which encode proteins interactingwith Bcl-2-family members. These methods include, for example, probingexpression libraries in a manner similar to the well known technique ofantibody probing of λgt11 libraries using labeled Bcl-2 relatedproteins, or a Bcl-2 related polypeptide, peptide or fusion protein.Protein-protein interactions can be measured using FluorescenceResonance Energy Transfer (FRET), e.g., by labeling Bcl-2-family membersand proteins that interact with Bcl-2-family members with suitable FRETprobes such as, but not limited to,5-(dimethylamino)naphthalene-1-sulfonyl chloride (dansyl chloride orDNS-Cl) attached to Tyr-69 andN-[[4-[4-(dimethylamino)phenyl]azo]phenyl]maleimide (DABMI) orN-[4-(dimethylamino)-3,5-dinitrophenyl]maleimide (DDPM). Alternately,two-hybrid systems can be used either to confirm protein-proteininteractions, or to search for previously unknown proteins capable ofbinding Bcl-2-family members.

[0192] The two-hybrid system for detecting protein-protein interactionsin vivo is described in detail for illustration only and not by way oflimitation. One version of this system has been described (Chien et al.,1991, Proc Natl Acad Sci USA 88:9578-9582) and is commercially available(Clontech, Palo Alto, Calif.). Briefly, plasmids are constructed thatencode two hybrid proteins: one plasmid consists of nucleotides encodingthe DNA-binding domain of a transcription activator protein fused to anucleotide sequence encoding the Bcl-2-family member, a polypeptide,peptide or fusion protein, and the other plasmid consists of nucleotidesencoding the transcription activator protein's activation domain fusedto a cDNA encoding an unknown protein which has been recombined intothis plasmid as part of a cDNA library. The DNA-binding domain fusionplasmid and the cDNA library are transformed into a strain of the yeastSaccharomyces cerevisiae that contains a reporter gene that expresses aprotein (e.g., HBS or lacZ) whose regulatory region contains the bindingsite of the transcription activator. Either hybrid protein alone cannotactivate transcription of the reporter gene: the DNA-binding domainhybrid cannot because it does not provide activation function and theactivation domain hybrid cannot because it cannot localize to theactivator's binding sites. Interaction of the two hybrid proteinsreconstitutes the functional activator protein and results in expressionof the reporter gene, which is detected by an assay for the reportergene product.

[0193] The two-hybrid system or related methodology may be used toscreen activation domain libraries for proteins that interact with the“bait” gene product. By way of example, and not by way of limitation,Bcl-2 may be used as the bait gene product. Total genomic or cDNAsequences are fused to the DNA encoding an activation domain. Thislibrary and a plasmid encoding a hybrid of a bait Bcl-2 gene productfused to the DNA-binding domain are cotransformed into a yeast reporterstrain, and the resulting transformants are screened for those thatexpress the reporter gene. For example, and not by way of limitation, abait Bcl-2 gene sequence can be cloned into a vector such that it istranslationally fused to the DNA encoding the DNA-binding domain of theGAL4 protein. These colonies are purified and the library plasmidsresponsible for reporter gene expression are isolated. DNA sequencing isthen used to identify the proteins encoded by the library plasmids.

[0194] A cDNA library of the cell line from which proteins that interactwith bait Bcl-2 related gene product are to be detected can be madeusing methods routinely practiced in the art. According to theparticular system described herein, for example, the cDNA fragments canbe inserted into a vector such that they are translationally fused tothe transcriptional activation domain of GAL4. This library can beco-transformed along with the bait Bcl-2 gene-GAL4 fusion plasmid into ayeast strain which contains a lacZ gene driven by a promoter whichcontains GAL4 activation sequence. A cDNA encoded protein, fused to GAL4transcriptional activation domain, that interacts with bait obR geneproduct will reconstitute an active GAL4 protein and thereby driveexpression of the HIS3 gene. Colonies which express HIS3 can be detectedby their growth on petri dishes containing semi-solid agar based medialacking histidine. The cDNA can then be purified from these strains, andused to produce and isolate the bait Bcl-2 gene-interacting proteinusing techniques routinely practiced in the art.

[0195] Similarly, TR3 and TCTP could serve as targets in the aboveassays to find interacting proteins and related compounds.

IX. Structure-Based Drug Design

[0196] To aid in the characterization and optimization of compoundswhich can alter the activity of Bcl-2-family proteins, structure-baseddrug design has become a useful tool. Solution nuclear magneticresonance (NMR) techniques can be used to map the interactions betweenthe BH3 domain of the Bcl-2-family protein and chemical compounds thattarget these anti-apoptotic proteins. NMR chemical shift perturbation isan efficient tool for rapid mapping of interaction interfaces onproteins. Structure-activity relationships (SAR) can be obtained byusing nuclear magnetic resonance (NMR), using the method known as “SARby NMR” (Shuker et al., 1996, Science 274:1531; Lugovskoy et al., 2002,J Am Chem Soc 124:1234). SAR by NMR can be used to identify, optimizeand link together small organic molecules that bind to proximal subsitesof a protein to produce high-affinity ligands.

[0197] In using NMR to structurally characterize protein-protein andligand-protein interactions, isotope labeling can result in increasedsensitivity and resolution, and in reduced complexity of the NMRspectra. The three most commonly used stable isotopes for macromolecularNMR are ¹³C, ¹⁵N and ²H. Isotope labeling has enabled the efficient useof heteronuclear multi-dimensional NMR experiments, providingalternative approaches to the spectral assignment process and additionalstructural constraints from spin-spin coupling. Uniform isotope labelingof the protein enables the assignment process through sequentialassignment with multidimensional triple-resonance experiments andsupports the collection of conformational constraints in de novo proteinstructure determinations (Kay et al., 1990 J Magn Reson 89:496; Kay etal., 1997, Curr Opin Struct Biol 7:722). These assignments can be usedto map the interactions of a ligand by following chemical-shift changesupon ligand binding. In addition, intermolecular NOE (nuclear Overhausereffect) derived inter-molecular distances can be obtained tostructurally characterize protein-ligand complexes.

[0198] In addition to uniform labeling, selective labeling of individualamino acids or labeling of only certain types of amino acids in proteinscan result in a dramatic simplification of the spectrum and, in certaincases, enable the study of significantly larger macromolecules. Forexample, the methyl groups of certain amino acids can be specificallylabeled with ¹³C and ¹H in an otherwise fully ²H-labeled protein. Thisresults in well resolved heteronuclear [¹³C,¹H]-correlation spectra,which enables straightforward ligand-binding studies either by chemicalshift mapping or by protein methyl-ligand inter-molecular NOEs, thusproviding key information for structure-based drug design in proteins aslarge as 170 kDa (Pellecchia et al., 2002, Nature Rev Drug Discovery1:211). 2D [¹³C, ¹H]-HMQC (heteronuclear multiple quantum coherence) and¹³C-edited [¹H,¹H]-NOESY NMR experiments on a ligand-receptor complexcan be used to detect binding, determine the dissociation constant forthe complex, and provide a low-resolution model based on the availablethree-dimensional structure of the target, thus revealing the relativeposition of the ligand with respect to labeled side-chains.

[0199] Thus, NMR can be used to identify molecules that induceapoptosis. Compounds can be screened for binding to labeled Bcl-X_(L),for example. Such labels include ¹⁵N and ¹³C. The interaction betweenthe compound and Bcl-X_(L), and therefore its ability to induceapoptosis, are determined via NMR.

X. Gene Therapy

[0200] Nucleic acid encoding TR3, TCTP, and deletions, truncations andvariations thereof, as well as any other peptides identified by themethods above may be used in gene therapy. In gene therapy applications,genes are introduced into cells in order to achieve in vivo synthesis ofa therapeutically effective product, for example the replacement of adefective gene. “Gene therapy” includes both conventional gene therapywhere a lasting effect is achieved by a single treatment, and theadministration of gene therapeutic agents, which involve the one time orrepeated administration of a therapeutically effective DNA or MRNA.Antisense RNAs and DNAs can be used as therapeutic agents for blockingthe expression of certain genes in vivo. It has been shown that shortantisense oligonucleotides can be imported into cells where they act asinhibitors, despite their low intracellular concentrations caused bytheir restricted uptake by the cell membrane. (Zamecnik et al., 1986,Proc Natl Acad Sci USA 83:4143-4146). The oligonucleotides can bemodified, e.g., by substituting their negatively charged phosphodiestergroups by uncharged groups.

[0201] There are a variety of techniques available for inducing nucleicacids into viable cells. The techniques vary depending upon whether thenucleic acid is transferred into cultured cells in vitro, or in vivo inthe cells of the intended host. Techniques suitable for the transfer ofnucleic acid into mammalian cells in vitro include, but are not limitedto, the use of liposomes, electroporation, microinjection, cell fusion.DEAE-dextran, and calcium phosphate precipitation method. The currentlypreferred in vivo gene transfer techniques include transfection withviral (typically retroviral)vectors and viral coat protein-liposomemediated transfection (Dzau et al., 1993, Trends in Biotechnology 11,205-210). In some situations it is desirable to provide the nucleic acidsource with an agent that targets the target cell, such as an antibodyspecific for a cell surface membrane protein or the target cell, or aligand for a receptor on the target cell. Where liposomes are employed,proteins which bind to a cell surface membrane protein associated withendocytosis may be used for targeting and/or to facilitate uptake, e.g.capsid proteins or fragments thereof that are tropic for a particularcell type, antibodies for proteins which undergo internalization incycling, and proteins that target intracellular localizations, andenhance intracellular half-life. The technique of receptor-mediatedendocytosis is described, e.g., by Wu et al., 1987, J Biol Chem 262:4429-4432 and Wagner et al., 1990, Proc Natl Acad Sci USA, 87:3410-3414.For review of gene therapy protocols see Anderson et al., 1992, Science256:808-813.

[0202] Given the teachings set forth herein, the skilled artisan mayselect among various vectors and other expression/delivery elementsdepending on such factors as the site and route of administration andthe desired level and duration of expression.

[0203] For example, naked plasmid DNA can be introduced into musclecells, for example, by direct injection into the tissue. (Wolff et al.,1989, Science 247:1465).

[0204] DNA-Lipid Complexes-Lipid carriers can be associated with nakedDNA (e.g., plasmid DNA) to facilitate passage through cellularmembranes. Cationic, anionic, or neutral lipids can be used for thispurpose. However, cationic lipids are preferred because they have beenshown to associate better with DNA which, generally, has a negativecharge. Cationic lipids have also been shown to mediate intracellulardelivery of plasmid DNA (Felgner and Ringold, 1989, Nature 337:387).Intravenous injection of cationic lipid-plasmid complexes into mice hasbeen shown to result in expression of the DNA in lung (Brigham et al.,1989, Am J Med Sci 298:278). See also, Osaka et al., 1996, J Pharm Sci85(6):612-618; San et al., 1993, Human Gene Therapy 4:781-788; Senior etal., 1991, Biochim Biophys Acta 1070:173-179; Kabanov and Kabanov, 1995,Bioconjugate Chem 6:7-20; Remy et al., 1994, Bioconjugate Chem5:647-654; Behr, 1994, Bioconjugate Chem 5:382-389; Behr et al., 1989,Proc Natl Acad Sci USA 86:6982-6986; and Wyman et al., 1977, Biochem36:3008-3017.

[0205] Adenovirus-based vectors for the delivery of transgenes are wellknown in the art and may be obtained commercially or constructed bystandard molecular biological methods. Recombinant adenoviral vectorscontaining exogenous genes for transfer are, generally, derived fromadenovirus type 2 (Ad2) and adenovirus type 5 (Ad5). They may also bederived from other non-oncogenic serotypes. See, e.g., Horowitz,“Adenoviridae and their Replication” in Virology, 2d Ed., Fields et al.eds., Raven Press Ltd., New York (1990) incorporated herein byreference.

[0206] It has been shown that TR3 and TCTP specifically interact withBcl-2-family receptors, resulting in the conversion of these moleculesfrom anti-apoptotic to pro-apoptotic. These studies provide a molecularbasis for developing various anti-cancer drugs and other therapeuticagents.

M. Methods of Identifying Antibodies which Induce a ConformationalChange in Bcl-2 Comparable to that Induced by TR3

[0207] Antibodies against the loop domain of Bcl-2. The N-terminal loopregion of Bcl-2 can be expressed and used as an antigen to developanti-Bcl-2/N-terminal loop region antibodies. These antibodies, bybinding to the loop domain of Bcl-2 can mimic the activity of TR3 in theinduction of a conformational change of Bcl-2 which will expose thehidden BH3 domain and confer pro-apoptotic activity to the Bcl-2protein. To identify antibodies that bind to the loop region, an ELISAassay can be used which is set up with various anti-loop antibodies forcapture and an anti-BH2 antibody for detection.

[0208] Assays to determine whether the antibody can mimic TR3 can be setup in a cellular system by transfecting the antibodies intoTR3-expression Bcl-2 deleted cells and looking for cellular effects.

[0209] These antibodies may be used to treat cancers or any diseases ofdecreased apoptosis or over-proliferation of cells. These antibodies mayalso be used to identify other therapeutic molecules which induce anequivalent conformational change to TR3. For example, compounds whichinhibit the interaction between the antibody and Bcl-2 may beidentified. Bcl-2 can be contacted with a candidate compound and aBH3-specific antibody (or any antibody which mimics TR3 by inducing aconformational change in Bcl-2). The contact may occur under conditionswhere the BH3 domain of Bcl-2 is not accessible to a BH3 specificantibody. The association of the BH3 specific antibody to the BH3 domainof Bcl-2 is then detected, whereby the candidate compound is identifiedas an agent that induces apoptosis or an agent that interferes withbinding of the BH3 specific antibody or an agent that allows theinteraction between Bcl-2 and the anti-BH3 specific antibody. Thus, theassay may be set up to identify a compound that interferes with thebinding of the antibody, or a compound that induces a conformationalchange allowing the antibody to bind.

[0210] Further, Bcl-X_(L) antibodies which mimic the activity of TCTPmay be identified in the same way and used for therapeutics.

XII. Methods of Diagnosis Using Antibodies which Recognize theConformational Change in Bcl-2 which is Induced by TR3

[0211] Antibodies described herein that are capable of binding to theconformationally changed Bcl-2 (for example to the conformationallyaccessible BH3 domain) can be used to identify the presence or absenceor amount of the Bcl-2 which is conformationally changed in patientsamples (the Bcl-2 epitope) and can be useful as diagnostics asdescribed hereinbelow in addition to their therapeutic effectiveness inthe treatment of malignant tissue growth and/or disease, such as cancerand obstructive tissue growths. In its simplest embodiment, such anantibody may be able to recognize the BH3 domain which is exposed by TR3binding.

[0212] An Enzyme-Linked Immunosorbent Assay (ELISA) for the detection ofthe epitope of Bcl-2 in a sample can be developed. In the assay, wellsof a microtiter plate, such as a 96-well microtiter plate or a 384-wellmicrotiter plate, are adsorbed for several hours with a first fullyhuman (for example) monoclonal antibody directed against the epitope.The immobilized antibody serves as a capture antibody for any of theantigen that can be present in a test sample. The wells are rinsed andtreated with a blocking agent such as milk protein or albumin to preventnonspecific adsorption of the analyte.

[0213] Subsequently the wells are treated with a test sample suspectedof containing the antigen, or with a solution containing a standardamount of the antigen. Such a sample can be, for example, a serum samplefrom a subject suspected of having levels of circulating antigenconsidered to be diagnostic of a pathology, for example, a cancer.

[0214] After rinsing away the test sample or standard, the wells aretreated with a second fully human monoclonal anti-Bcl-2 antibody that islabeled by conjugation with biotin. The labeled anti-Bcl-2 antibodyserves as a detecting antibody. After rinsing away excess secondantibody, the wells are treated with avidin-conjugated horseradishperoxidase (HRP) and a suitable chromogenic substrate. The concentrationof the antigen in the test samples is determined by comparison with astandard curve developed from the standard samples.

[0215] This ELISA assay provides a highly specific and very sensitiveassay for the detection of the Bcl-2 epitope (which mimics theconformation change induced by TR3) in a test sample.

[0216] For determination of the epitope of Bcl-2 in patients, a sandwichELISA is developed which works with human serum and/or tissue samplesfrom the patient. The two fully human monoclonal anti-bcl-2 antibodiesused in the sandwich ELISA, recognizes different epitopes on the bcl-2molecule. Normal or patient sera can be diluted in blocking buffer. Thismethod can be used for diagnosing the presence of a cancer or staging ofcancer in a patient based on the amount of the conformationally inducedBcl-2 present.

[0217] To develop the assay for a given type of cancer, samples of bloodare taken from subjects diagnosed as being at various stages in theprogression of the disease, and/or at various points in the therapeutictreatment of the cancer. The concentration of the specific Bcl-2 epitopeor antigen present in the blood samples is determined using a methodthat specifically determines the amount of the antigen that is present.Such a method includes an ELISA method. This may also be used to stagethe progression of the cancer in a subject under study, or tocharacterize the response of the subject to a course of therapy.

[0218] Further antibodies which recognize the conformationally changedBcl-X_(L) can be used for diagnosis and prognosis in the same way asthose for Bcl-2 above.

What is claimed is:
 1. A compound which binds to Bcl-2 and modulates theactivity of Bcl-2 in a cell so as to be inductive of apoptosis, andwherein the compound does not cleave Bcl-2.
 2. The compound of claim 1wherein said compound induces a conformational change in Bcl-2.
 3. Thecompound of claim 1 wherein said compound is selected from the groupconsisting of a peptide, peptidomimetic, an antibody or part thereof anda small organic molecule.
 4. The compound of claim 1, wherein thecompound comprises TR3.
 5. The compound of claim 1, wherein the compoundcomprises the DC1 region of TR3.
 6. A compound which binds to Bcl-XL andmodulates the activity of Bcl-XL in a cell so as to be inductive ofapoptosis, and wherein the compound does not cleave Bcl-XL.
 7. Thecompound of claim 5 wherein said compound is selected from the groupconsisting of a peptide, peptidomimetic, an antibody or part thereof anda small organic molecule.
 8. The compound of claim 5 wherein thecompound comprises TCTP.
 9. The compound of claim 5 wherein the compoundcomprises a peptidomimetic mimicking TCTP.
 10. A method of inducingapoptosis in a mammalian cell, comprising contacting said cell with aneffective amount of a compound which binds to Bcl-2 and modulates theactivity of Bcl-2 in said cell so as to be inductive of apoptosis.
 11. Amethod of inducing apoptosis in a mammalian cell, comprising contactingsaid cell with an effective amount of a compound which binds to Bcl-XLand modulates the activity of Bcl-XL in said cell so as to be inductiveof apoptosis.
 12. A method of inhibiting apoptosis in a mammalian cell,comprising binding contacting said cell with an effective amount of acompound that prevents the binding of TR3 and Bcl-2.
 13. The method ofclaim 11, wherein said compound comprises a peptide, peptide analogue,or small molecule designed to block association between TR3 and Bcl-2.14. A method of inhibiting apoptosis in a mammalian cell, comprisingcontacting said cell with an effective amount of a compound thatprevents the binding of TCTP and Bcl-XL.
 15. The method of claim 13,wherein said compound is selected from the group consisting of: apeptide, a peptide analogue, an antibody or part thereof and a smallmolecule designed to block association between TCTP and Bcl-XL.
 16. Themethod of claim 13, wherein said compound comprises TCTP antisense RNA.17. A method of identifying molecules that inhibit apoptosis, comprisingmeasuring the amount of labeled TCTP or TR3 bound to Bcl-2 familyproteins anchored to a solid support in the presence and absence ofmolecules being tested, and determining the ability of each saidmolecule being tested to compete with TCTP or TR3 for binding sites onBcl-2 family proteins.
 18. A method of identifying molecules that induceapoptosis, comprising determining the ability of said molecule to bindto a Bcl-2-family protein and modulate the activity of said protein soas to be inductive of apoptosis.
 19. A method of identifying moleculesthat induce apoptosis, comprising screening compounds using NMR forbinding to N15-Bcl-2 at the same site where TR3 binds, said site beingdifferent from the BH3-binding site on Bcl-2.
 20. The method of claim17, additionally comprising 13C labeling of Bcl-2.
 21. A method ofidentifying molecules that induce apoptosis, comprising screeningcompounds using NMR for binding to N15-Bcl-XL at the same site whereTCTP binds, said site being different from the BH3-binding site onBcl-XL.
 22. The method of claim 17, additionally comprising 13C labelingof Bcl-XL.
 23. A method for identifying molecules that induce apoptosis,comprising: (a) detecting a labeled Bcl-2 binding compound bound toBcl-2, wherein said Bcl-2 binding compound is known to induce aconformational change in Bcl-2 so as to be inductive of apoptosis; (b)contacting the Bcl-2 binding compound—Bcl-2 complex with a candidateagent, the candidate agent suspected of being able to induce aconformational change in Bcl-2 so as to be inductive of apoptosis, and(c) detecting dissociation of the labeled Bcl-2 binding compound fromthe complex, whereby the candidate compound is identified as an agentthat induces apoptbsis.
 24. The method of claim 21 wherein said Bcl-2binding compound is selected from the group consisting of: TR3, theligand binding domain of TR3, an antibody that mimics TR3, a peptidecomprising the DC1 region of TR3, a functional fragment of TR3, and apeptidomimetic.
 25. The method of claim 21 wherein the Bcl-2 protein isa fragment of the Bcl-2 protein comprising the BH3 and BH4 domains. 26.The method of claim 21 wherein the Bcl-2 protein is a fragment of theBcl-2 protein comprising the N-terminal loop region, located between theBH4 and BH3 domains.
 27. The method of claim 21 wherein the method iscarried out using SAR by NMR.
 28. The method of claim 21 wherein themethod is carried out using high throughput screening.
 29. A method foridentifying molecules that induce apoptosis, comprising: (a) detecting alabeled Bcl-X_(L) binding compound bound to Bcl-X_(L), wherein saidBcl-X_(L) binding compound is known to induce a conformational change inBcl-X_(L) so as to be inductive of apoptosis; (b) contacting theBcl-X_(L) binding compound—Bcl-X_(L) complex with a candidate agent, thecandidate agent suspected of being able to induce a conformationalchange in Bcl-X_(L) so as to be inductive of apoptosis, and (c)detecting dissociation of the labeled Bcl-X_(L) binding compound fromthe complex, whereby the candidate compound is identified as an agentthat induces apoptosis.
 30. The method of claim 21 wherein saidBcl-X_(L) binding compound is selected from the group consisting of:TCTP or a functional fragment thereof, an antibody which mimics theaction of TCTP, and a peptidomimetic.
 31. A method for identifyingmolecules that induce apoptosis, comprising: (a) contacting Bcl-2 with acandidate compound in the presence of a multidomain pro-apoptoticBcl-2-family protein, and (b) detecting the association of Bcl-2 withsuch multidomain pro-apoptotic Bcl-2-family protein, whereby thecandidate compound is identified as an agent that induces apoptosis. 32.A method for identifying molecules that induce apoptosis, comprising:(a) contacting Bcl-X_(L) with a candidate compound in the presence of amultidomain pro-apoptotic Bcl-2-family protein, and (b) detecting theassociation of Bcl-X_(L) with such multidomain pro-apoptoticBcl-2-family protein, whereby the candidate compound is identified as anagent that induces apoptosis.
 33. A method for identifying moleculesthat induce apoptosis, comprising: (a) contacting Bcl-2 with a candidatecompound and a BH3 specific antibody under conditions where the BH3domain of Bcl-2 is not accessible to a BH3 specific antibody, and (b)detecting the association of the BH3 specific antibody to the BH3 domainof Bcl-2, whereby the candidate compound is identified as an agent thatinduces apoptosis.
 34. A method of inducing apoptosis in a cellcomprising administering a compound capable of inducing aconformnational change in Bcl-2 so as to be inductive of apoptosis. 35.The method of claim 32 wherein the compound works through inducing TR3to localize to the mitochondria of the cell.
 36. A method of inducingapoptosis in a mammalian cell, comprising contacting said cell with aneffective amount of a compound which enhances the binding of TR3 toBcl-2 in said cell so as to be inductive of apoptosis.
 37. An antibodyspecific for the BH3 domain of Bcl-2 which can be used to determinewhether Bcl-2 has undergone a conformational change so as to beinductive of apoptosis.